改进的类异戊二烯生产的制作方法

文档序号:603010阅读:1163来源:国知局
专利名称:改进的类异戊二烯生产的制作方法
技术领域
本发明涉及新的用于类异戊二烯生物合成途径的多核苷酸和多肽序列。更具体地,本发明涉及重组制备的细胞,该细胞显示出改进的玉米黄质产量。还提供了制备和使用这种细胞系的方法。
类胡萝卜素是一种重要的C-40类异戊二烯化合物商品,可作为人类的营养性支持物、药物和食用色素,还可作为动物饲料的色素。目前重要的工业类胡萝卜素的生产主要是通过化学合成(β-胡萝卜素,角黄素和虾青素)或者从天然材料中提取(叶黄素提取自万寿菊,辣椒红提取自红辣椒(paprika))。但是,使用微生物生产类胡萝卜素已经取得了几例成功。例如,使用真菌三孢布拉霉(blakeslea trispora)(US 5,328,845)进行发酵或使用耐盐藻类Dunaliella salina进行池塘养殖[Borowitzka,J.Biotechnol.70313-321(1999)]都可生产β-胡萝卜素。还有三孢布拉霉(B.trispora)生产番茄红素的报道(WO 00/77234)。使用酵母(Phaffia rhodozyma,(最近更名为Xanthophyllomyces dendorous))(US 6,015,684)发酵或使用藻类Haematococuspluvialis在光生物反应器内或开放的池塘内可以生产虾青素[Lorenz和Cysewski,Trends Biotechnol.18160-167(1999);Olaizola,J.Appl.Phycol.12499-506(2000)]。但是这种微生物生产系统生产的类胡萝卜素数量过低,工业规模生产则难以产生经济效益。
在1960年代中期,Hoffmann-La Roche的科学家们分离了几种海洋细菌,其能够生产黄色的类胡萝卜素玉米黄质,该物质可用于家禽色素形成以及预防人类中与年龄相关的黄斑变性。一种细菌,其显示出极有潜力的玉米黄质产率,被命名为R-1512,已保藏在美国典型培养物保藏中心(American Type Culture Collection)(ATCC,Manassas,VA,USA),保藏号为ATCC 21588(US 3,891,504)。使用当时公认的分类标准(按照EidgenossischeTechnische Hochschule(Zurich)和National Collection of Industrial Bactria,Torry Research Station(Aberdeen Scotland)进行的分类),这个能够生产玉米黄质的有机体被归类为黄杆菌属(Flavobacterium),但是没有命名种名。
然后进行了大量的诱变和筛选过程,以分离得到具有更高玉米黄质产率的R-1512突变体。在本文描述的研究中,有两种这样的突变体比较显著。按照玉米黄质产率顺序,列出这两个突变体R1534和R114。在过去几年中还用过各种其它的突变体,用于类胡萝卜素的生物合成的生化研究[Goodwin,Biochem.Soc.Symp.35233-244(1972);McDermott等,Biochem.J.1341115-1117(1973);Britton等,Arch.Microbiol.11333-37(1977);Mohanty等,Helvetica Chimica Acta 832036-2053(2000)]。
通过常规利用R-1512菌株衍生的突变体发展一种商业生命力的发酵过程来生产玉米黄质的早期尝试没有成功。但是,随着分子生物学的进展,发展一种更高玉米黄质产率菌株的可能性随之出现。为此,采取的第一个步骤是克隆并测序R-1534菌株的类胡萝卜素基因簇(US 6,087,152,将该文献全文并入作为参考)。US 6,087,152公开了类胡萝卜素基因在大肠杆菌和枯草芽孢杆菌(Bacillus Subtilis)中功能性表达,使得这些宿主产生玉米黄质。US 6,087,152还公开了通过修饰类胡萝卜素基因簇或加入自产虾青素细菌的基因,就有可能产生玉米黄质之外的各种类胡萝卜素(EP 872,554)。而且,EP 872,554公开了通过向多拷贝质粒导入克隆的类胡萝卜素基因簇,可以提高R1534菌株的类胡萝卜素产量。
尽管类异戊二烯化合物存在大量的结构多样性,但是都是由一个共同的C-5前体,异戊烯焦磷酸(IPP)生物合成而来的。直到1990年代早期,才基本上接受了IPP是通过甲羟戊酸途径在所有有机体内合成,即使有些实验结果与这个生物发生模式不一致[Eisenreich等,Chemistry and Biology5R211-R233(1998)]。
甲羟戊酸途径
自从发现了IPP生物合成的替代性途径,脱氧木糖醇(DXP)途径之后,这种不一致就找到了解释(注释在科学文献中,IPP生物合成的替代性途径已被命名了多个名字(DXP途径,DOXP途径,MEP途径,GAP/丙酮酸途径以及非甲羟戊酸途径。本文中为简化,仅使用DXP途径)。DXP途径的前5个反应已被鉴定[Herz等,Proc.Nat.Acad.Sci 972486-2490(2000)],但是其后生成IPP的步骤尚不清楚。
DXP途径
McDermott等(同上)和Britton等[J.Chem.Soc.Chem.Comm.p.27(1979)]揭示源自原Roche分离群的产玉米黄质突变株,其粗提物中的玉米黄质掺入了标记的甲羟戊酸。没有理由再质疑这个通过甲羟戊酸途径进行IPP生物合成的证据,这项研究是在发现DXP途径之前进行的,已有报道称一些细菌(链霉菌属的种类)同时具备IPP合成的这两种途径,并且这些途径的表达受时间(temporally)调控[Seto等,Tetrahedron Lett.377979-7982(1996);Dairi等,Mol.Gen.Genet.262957-964(2000)]。另外,目前只有少数的真细菌显示具有IPP合成的甲羟戊酸途径。这些细菌中编码甲羟戊酸途径各种酶的基因已被克隆并测序[Wilding等,J.Bacteriol.1824319-4327(2000);Takagi等,J.Bacteriol 1824153-4157(2000)]。
已有几个范例显示代谢工程的应用已成功地改变或改进了微生物生产类胡萝卜素的产量[Lagarde等,Appl.Env.Microbiol.6664-72(2000);Wang等,Biotechnol.Bioeng.62235-241(1999);Wang等,Biotechnol. Prog.16922-926(2000)(作为本文的参考);Sandmann等,Trends Biotechnol.17233-237(2000);Misawa and Shimada,J.Biotechnol.59-169-181(1998);Matthews and Wurtzel,Appl.Microbiol.Biotechnol.53396-400(2000);Albrecht et al.,Nature Biotechnol.18843-846(2000);Schmidt-Dannert et al.Nature Biotechnol.18750-753(2000)]。例如,大肠杆菌,不具备胡萝卜素形成作用(non-carotenogenic)的细菌,可通过导入克隆的类胡萝卜素(crt)基因进行改造使细菌生产类胡萝卜素,所述细菌Agrobacterium aurantiacum,草生欧文氏菌(Erwinia herbicola)或菠萝欧文氏菌(Erwinia uredovora)(Misawa和Shimada,同上)。Harker和Bramley[FEBS Lett.448115-119(1999)]和Matthews和Wurtzel(同上)公开通过过量表达编码1-脱氧-D-木糖醇-5-磷酸合成酶(DXPS)的基因,可提高这种改造过的大肠杆菌菌株中的类胡萝卜素产量,该酶是DXP途径中的第一个酶(大肠杆菌只有类异戊二烯生物合成的DXP途径,并不使用甲羟戊酸途径[Lange et al.,Proc.Nat.Acad.Sci.9713172-13177(2000)])。Harker和Bramley(同上)还公开了在过量产生DXPS的细胞中,类异戊二烯化合物泛醌-8的升高。这些结果均支持如下的假说由于DXPS体内活性不足,导致IPP有效性受限,这就限制了类胡萝卜素以及其它类异戊二烯化合物在改造菌株中的产量。使用类似的大肠杆菌体系,Kim和Keasling[Biotechnol.Bioeng.72408-415(2001)]公开了组合的过量表达编码DXPS和DXP途径的第二个酶——DXP还原异构酶(1-脱氧-D-木酮糖-5-磷酸还原异构酶)(1-deoxy-D-xylulose-5-phosphate reductoisomerase)的基因,所得到的类胡萝卜素产量高于仅仅过量表达编码DXPS的基因的产量。
所有这些研究都是对大肠杆菌进行改造使其生产类胡萝卜素。相应地,这些研究的一个不足之处在于这些重组大肠杆菌菌株生产的类胡萝卜素产量即使跟工业化生产类胡萝卜素的非重组的微生物相比,也非常低。另外,通过遗传工程改造IPP生物合成途径在细菌中改进类胡萝卜素生产只在应用DXP途径进行IPP合成的生物体中才能看到。对于经甲羟戊酸途径生成IPP的细菌则没有类似的研究报道。
已经报道了在酵母中通过甲羟戊酸的代谢工程以改进类异戊二烯化合物的生产。例如,WO 00/01649公开了当3-羟基-3-甲基戊二酸单酰辅酶A还原酶(HMG-CoA还原酶)被过量表达时,类异戊二烯化合物在酿酒酵母(Saccharomyces cerevisiae)中的产量有所提高。但是,未显示该方案能够改进细菌中类胡萝卜素的产量,特别是它没有显示出通过扩增甲羟戊酸途径各基因的表达可以改进细菌中类胡萝卜素的产量。已经发现,一些真核生物[Campos等,Biochem.J.35359-67(2001)]和链霉菌属细菌菌株CL190(Takagi等,同上)的甲羟戊酸途径基因可以在大肠杆菌中表达,未报道在这些菌株中类异戊二烯的产量有所提高。
除了形成IPP[通过DXP或甲羟戊酸途径]以及将法呢基焦磷酸转换为各种其它类异戊二烯(如类胡萝卜素,苯醌)的反应之外,还已知两种反应涉及类异戊二烯的生物合成。IPP异构酶使IPP及其异构体——二甲基烯丙基焦磷酸(DMAPP)两者之间相互转变。存在两种形式的IPP异构酶,众所周知真核生物和一些细菌中的1型酶,而新近鉴定的2型酶是FMN-和NADP(H)依赖性的[Kaneda等,Proc.Nat.Acad.Sci.98932-937(2001)]。
几篇报道公开了在改造的产类胡萝卜素大肠杆菌中,扩增天然或异源的1型IPP异构酶(idi)基因可以促进类胡萝卜素的产量[Kajiwara等,Biochem.J.324421-426(1997);Verdoes和van Ooyen,Acta Bot.Gallica 14643-53(1999);Wang等,同上]。在一篇报道(Wang等,同上)中进一步公开当idi和crtE(GGPP合成酶/geranyl-geranyl diphosphate synethase)基因组合过量表达时,过量表达编码FPP合成酶(法呢基二磷酸合成酶(farnesyldiphosphate synthase))的ispA基因,可提高改造的产类胡萝卜素大肠杆菌菌株的类胡萝卜素产量。但是,对于IPP生物合成途径而言,没有显示出过量表达编码IPP异构酶或FPP合成酶的基因可以改进天然产类胡萝卜素微生物的类胡萝卜素产量。同样,上述的大肠杆菌菌株所产生的类胡萝卜素水平很低,而且,在原本类胡萝卜素产量较高的工业微生物中,这种策略还未显示出作用。
总之,没有任何现有证据证明提高编码甲羟戊酸途径的各酶基因的表达可以改进天然产类胡萝卜素(carotenogenic)细菌、或天然不生产类胡萝卜素而被改造使其生产类胡萝卜素的细菌的类胡萝卜素产量。
本发明的一个实施方案是分离的多肽,其含有选自下列的氨基酸序列(a)SEQ ID NO43中1-340残基所示的氨基酸序列;(b)SEQ ID NO45中1-349残基所示的氨基酸序列;(c)SEQ ID NO47中1-388残基所示的氨基酸序列;(d)SEQ ID NO49中1-378残基所示的氨基酸序列;(e)SEQ ID NO51中1-305残基所示的氨基酸序列;(f)SEQ ID NO53中1-332残基所示的氨基酸序列;(g)选自SEQ ID NO43,45,47,49,51和53所示氨基酸序列的片段,其中所述片段至少具有30个连续的氨基酸残基;(h)选自SEQ ID NO43,45,47,49,51和53所示多肽片段的氨基酸序列,所述片段具有HMG-CoA还原酶,异戊烯二磷酸异构酶,羟基甲基戊二酸单酰-CoA合成酶(HMG-CoA合成酶),甲羟戊酸激酶,磷酸甲羟戊酸激酶,或二磷酸甲羟戊酸脱羧酶的活性;(i)严谨条件下与杂交探针杂交的多核苷酸所编码的多肽氨基酸序列,所述杂交探针含有SEQ ID NO42或其互补序列的至少30个连续核苷酸,其中所述多肽具有HMG-CoA还原酶,异戊烯二磷酸异构酶,HMG-CoA合成酶,异戊烯二磷酸异构酶,甲羟戊酸激酶,磷酸甲羟戊酸激酶,或二磷酸甲羟戊酸脱羧酶的活性;以及(j)SEQ ID NO43,45,47,49,51或53经保守修饰而形成的变体。
如上所述,本发明包括SEQ ID NO43,45,47,49,51和53,其分别相应于下述甲羟戊酸途径各酶的多肽序列羟甲基戊二酸单酰CoA(HMG-CoA)(hydroxymethyl glutaryl CoA)还原酶,异戊烯二磷酸(IPP)异构酶,HMG-CoA合成酶,甲羟戊酸激酶,磷酸甲羟戊酸激酶和二磷酸甲羟戊酸脱羧酶。本发明还包括每个鉴定序列的至少30个连续氨基酸,或足够数目的连续氨基酸,以定义生物活性分子。
本发明还包括选自SEQ ID NO43,45,47,49,51和53多肽的片段。该片段的长度至少应约为30个氨基酸长,且必须具有鉴定多肽的活性,例如对于SEQ ID NO43而言,其落入本发明范围之内的片段具有HMG-CoA还原酶的活性。在本文中,各片段活性的测定在实施例1中描述。实施例1所述的试验中具有高于背景活性的片段被认为具有生物学活性,属于本发明的范围。
本发明还包括多核苷酸所编码多肽的氨基酸序列,所述多核苷酸在严谨条件(如上面所定义)下能够与杂交探针杂交,所述探针含有SEQ ID NO42(即甲羟戊酸操纵子)或其互补序列的至少30个连续核苷酸。该多核苷酸必须编码甲羟戊酸途径的至少一种酶。本发明中,“杂交探针”指含有SEQID NO42的大约10-9066核苷酸的多核苷酸序列。
在该实施方案中,分离的多肽可以具有SEQ ID NO43,SEQ ID NO45,SEQ ID NO47,SEQ ID NO49,SEQ ID NO51或SEQ ID NO53的氨基酸序列。或者,该分离的多肽可含有选自各个氨基酸序列区域的、大约30个连续的氨基酸,与来自不同种类,且具有相同功能的酶相比,所述氨基酸序列具有最小的同源性。因此,本发明多肽可含有例如SEQ ID NO43的68-97,SEQ ID NO45的1-30,SEQ ID NO47的269-298,SEQ ID NO49的109-138,SEQ ID NO51的198-227或SEQ ID NO53的81-110氨基酸。
本发明的另一实施方案是分离的多肽,其具有选自下述的氨基酸序列(a)SEQ ID NO159的1-287残基所示的氨基酸序列;(b)SEQ ID NO159的至少30个连续的氨基酸残基;(c)SEQ ID NO159的片段的氨基酸序列,所述片段具有法呢基-二磷酸合成酶(FPP合成酶)的活性;(d)在严谨条件下与杂交探针杂交的多核苷酸所编码的多肽氨基酸序列,所述杂交探针含有ispA基因(即SEQ ID NO157的295-1158核苷酸)或其互补序列的至少30个连续核苷酸,其中所述多肽具有FPP合成酶的活性;以及(e)SEQ ID NO159经保守修饰而形成的变体。
这样,该实施方案中,氨基酸可由编码FPP合成酶的整个开放阅读框架编码,即SEQ ID NO159的1-287残基,其至少30个连续的残基,或SEQ ID NO159的片段,其必须具有FPP合成酶活性,如实施例1中所描述的试验所测定。另外,本发明的该实施方案还包括在严谨条件(如上定义)下,能够与杂交探针杂交的多核苷酸所编码的氨基酸序列,所述探针含有ispA基因(即SEQ ID NO157的295-1158核苷酸)或其互补序列的至少30个连续核苷酸,其中所述多肽如上定义具有FPP合成酶的活性。
在一个优选实施方案中,该多肽具有SEQ ID NO159的氨基酸序列。
本发明的另一实施方案是分离的多肽,其具有选自下述的氨基酸序列(a)SEQ ID NO160的1-142残基所示的氨基酸序列;(b)SEQ ID NO160的至少30个连续的氨基酸残基;(c)SEQ ID NO160的片段的氨基酸序列,所述片段具有1-脱氧木糖醇-5-磷酸合成酶(DXPS)的活性;(d)在严谨条件下能够与杂交探针杂交的多核苷酸所编码的多肽氨基酸序列,所述杂交探针含有SEQ ID NO157或其互补序列1185-1610位的至少30个连续核苷酸,其中所述多肽具有DXPS合成酶的活性;以及(e)SEQ ID NO160经保守修饰而形成的变体。
这样,该实施方案中,氨基酸可由编码DXPS合成酶的整个开放阅读框架编码,即SEQ ID NO160的1-142残基,其至少30个连续的残基,或SEQ ID NO160的片段,其如实施例1中所描述的试验所测定具有DXPS合成酶活性。另外,本发明的该实施方案还包括在严谨条件(如上定义)下,能够与杂交探针杂交的多核苷酸所编码的氨基酸序列,所述探针含有DXPS基因(即SEQ ID NO157的1185-1610核苷酸)或其互补序列的至少30个连续核苷酸,其中所述多肽如上定义具有DXPS合成酶的活性。
在一个优选实施方案中,该多肽具有SEQ ID NO160的氨基酸序列。
本发明的另一实施方案是分离的多肽,其具有选自下述的氨基酸序列(a)SEQ ID NO178的1-390残基所示的氨基酸序列;(b)SEQ ID NO178的至少30个连续的氨基酸残基;(c)SEQ ID NO178的片段的氨基酸序列,所述片段具有乙酰-CoA乙酰基转移酶的活性;(d)在严谨条件下能够与杂交探针杂交的多核苷酸所编码的多肽氨基酸序列,所述杂交探针含有phaA基因(即SEQ ID NO177的1-1179核苷酸)或其互补序列的至少30个连续核苷酸,其中所述多肽具有乙酰-CoA乙酰基转移酶的活性;以及(e)SEQ ID NO178经保守修饰而形成的变体。
这样,该实施方案中,氨基酸可由编码乙酰-CoA乙酰基转移酶的整个开放阅读框架编码,即SEQ ID NO178的1-143残基,其至少30个连续的残基,或SEQ ID NO178的片段,其必须具有乙酰-CoA乙酰基转移酶活性,如实施例1中所描述的试验所测定。另外,本发明的该实施方案还包括在严谨条件(如上定义)下,能够与杂交探针杂交的多核苷酸所编码的氨基酸序列,所述探针含有phaA基因(即SEQ ID NO177的1-1170核苷酸)或其互补序列的至少30个连续核苷酸,其中所述多肽如上定义具有乙酰-CoA乙酰基转移酶的活性。
在一个优选实施方案中,该多肽具有SEQ ID NO178的氨基酸序列。
本发明的另一实施方案是分离的多肽,其具有选自下述的氨基酸序列(a)SEQ ID NO179的1-240残基所示的氨基酸序列;(b)SEQ ID NO179的至少30个连续的氨基酸残基;(c)SEQ ID NO179的片段的氨基酸序列,所述片段具有乙酰乙酰-CoA还原酶的活性;(d)在严谨条件下能够与杂交探针杂交的多核苷酸所编码的多肽氨基酸序列,所述杂交探针含有phaB基因(即SEQ ID NO177的1258-1980核苷酸)或其互补序列的至少30个连续核苷酸,其中所述多肽具有乙酰乙酰-CoA还原酶的活性;以及(e)SEQ ID NO179经保守修饰而形成的变体。
这样,该实施方案中,氨基酸可由编码乙酰乙酰-CoA还原酶的整个开放阅读框架编码,即SEQ ID NO179的1-240残基,其至少30个连续的残基,或SEQ ID NO179的片段,该片段具有乙酰乙酰-CoA还原酶活性,如实施例1中所描述的试验所测定。另外,本发明的该实施例还包括在严谨条件(如上定义)下,能够与杂交探针杂交的多核苷酸所编码的氨基酸序列,所述探针含有phaB基因(即SEQ ID NO177的1258-1980核苷酸)或其互补序列的至少30个连续核苷酸,其中所述多肽如上定义具有乙酰乙酰-CoA还原酶的活性。
在一个优选实施方案中,该多肽具有SEQ ID NO179的氨基酸序列。
本文所用的术语“多肽”,“多肽序列”,“氨基酸”和“氨基酸序列”可互换使用,指寡肽,肽,多肽,或蛋白序列,或它们的片段,以及天然存在或合成的分子。在本文中,“片段”,“免疫原性片段”,或“抗原性片段”指本文所定义的任一多肽的片段,该片段长度至少约为30个氨基酸,并具有所述多肽的某些生物活性或免疫活性。当用“氨基酸序列”描述天然蛋白分子的氨基酸序列时,“氨基酸序列”及其类似的术语并不是将氨基酸序列限定为与所述的蛋白分子相关的完全天然的氨基酸序列。
当用于描述多肽时,术语“分离的”指蛋白或多肽已经从天然状态下与其混合(accompany)的各个组分中分离出来。当样品的至少约60-75%呈现为单一多肽序列时,即分离得到了单体蛋白。分离的蛋白通常含有约60-90%W/W的蛋白样品,更通常约为95%,优先高于约99%纯度。蛋白纯度或均一性可利用本领域公知的许多方式表示,如蛋白样品的聚丙烯酰胺凝胶电泳,再通过染色凝胶观测单一多肽条带。为满足某些目的,可使用HPLC或其它本领域公知的方法提供更高的纯化分辨率。
本文中术语“生物活性”指具有天然存在分子的结构,调节性或生化功能的蛋白。同样,“免疫活性”指天然,重组或合成多肽或其任一寡肽的性能,该性能可在适当的动物或细胞内诱导特定免疫应答,并结合特定抗体。
本发明的另一实施方案是分离的多核苷酸序列,其具有甲羟戊酸操纵子(SEQ ID NO42)的核苷酸序列,SEQ ID NO42各变体的核苷酸序列,所述变体含有一个或多个符合副球菌种(Paracoccus sp.)菌株1534密码子利用表(参见表14)的取代,或SEQ ID NO42的片段。SEQ ID NO42的变体和片段必须编码具有选自下述活性的多肽HMG-CoA还原酶,异戊烯二磷酸异构酶活性,羟基甲基戊二酸单酰-CoA合成酶(HMG-CoA合成酶),甲羟戊酸激酶,磷酸甲羟戊酸激酶和二磷酸甲羟戊酸脱羧酶。该实施方案还包括在如上定义的严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针的核苷酸序列由SEQ ID NO42的大约10到约9066个核苷酸构成,优选由SEQ ID NO42或其互补序列的30个连续核苷酸构成,该多核苷酸编码具有选自下述活性的多肽HMG-CoA还原酶,异戊烯二磷酸异构酶,HMG-CoA合成酶,甲羟戊酸激酶,磷酸甲羟戊酸激酶和二磷酸甲羟戊酸脱羧酶。
该实施方案还包括分离的多核苷酸序列,其含有SEQ ID NO42的下述残基2622-3644,3641-4690,4687-5853,5834-6970,6970-7887,7880-8878。这些序列的片段也包括在本发明的范围之内,只要是这些片段能够分别编码具有HMG-CoA还原酶活性,异戊烯二磷酸异构酶活性,HMG-CoA合成酶活性,甲羟戊酸激酶活性,磷酸甲羟戊酸激酶活性和二磷酸甲羟戊酸脱羧酶活性的多肽。
该实施方案还包括在如上定义的严谨条件下,能够与杂交探针杂交的多核苷酸序列,所述探针选自下述由SEQ ID NO422622-3644,3641-4690,4687-5853,5834-6970,6970-7887,7880-8878或其互补序列的至少30个连续核苷酸残基所构成的核苷酸序列,其中多核苷酸分别编码选自具有HMG-CoA还原酶活性,异戊烯二磷酸异构酶活性,HMG-CoA合成酶活性,甲羟戊酸激酶活性,磷酸甲羟戊酸激酶活性或二磷酸甲羟戊酸脱羧酶活性的多肽。
优选地,该分离的多核苷酸由SEQ ID NO42的2622-3644,3641-4690,4687-5853,5834-6970,6970-7887,7880-8878核苷酸构成。
本发明的另一实施方案是分离的多核苷酸序列,其具有SEQ ID NO157的核苷酸序列,SEQ ID NO157各变体的核苷酸序列,所述变体含有一个或多个符合副球菌种菌株1534密码子利用表(参见表14)的取代,或SEQID NO157的片段,SEQ ID NO157的变体和片段均编码具有下述活性的多肽FPP合成酶活性,1-脱氧-D-木酮糖-5-磷酸合成酶活性或XseB活性。该实施方案还包括在如上定义的严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针的核苷酸序列由SEQ ID NO157或SEQ ID NO157的互补序列的至少30个连续核苷酸构成,其中该多核苷酸编码具有下述活性的多肽FPP合成酶活性,1-脱氧-D-木酮糖-5-磷酸合成酶活性或XseB活性。
优选地,该分离的多核苷酸由SEQ ID NO157的59-292,295-1158或1185-1610位核苷酸构成。
还提供了具有选自下述核苷酸序列的分离多核苷酸序列SEQ ID NO157的59-292位核苷酸,SEQ ID NO157各变体的核苷酸序列,所述变体含有一个或多个符合副球菌种菌株R1534密码子利用表(参见表14)的取代,SEQ ID NO157的59-292位核苷酸序列的片段,该片段编码具有XseB功能的多肽,以及在严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针的核苷酸序列由SEQ ID NO157或其互补序列的59-292位的至少30个连续核苷酸构成,其中该多核苷酸编码具有XseB功能的多肽。
优选地,该分离的多核苷酸由SEQ ID NO157的59-292位核苷酸构成。
还提供了具有选自下述核苷酸序列的分离多核苷酸序列SEQ ID NO157的295-1158位核苷酸,SEQ ID NO157的295-1158位核苷酸序列的各变体,所述变体含有一个或多个符合副球菌种菌株R1534密码子利用表(参见表14)的取代,SEQ ID NO157的295-1158位核苷酸序列的片段,该片段编码FPP合成酶活性,以及在严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针的核苷酸序列由SEQ ID NO157或其互补序列的295-1158位的至少30个连续核苷酸构成,其中该多核苷酸编码具有FPP合成酶活性的多肽。
优选地,该分离的多核苷酸由SEQ ID NO157的295-1158位核苷酸构成。
本发明的另一实施方案是具有SEQ ID NO157的1185-1610位核苷酸序列的分离多核苷酸序列,SEQ ID NO157的1185-1610位核苷酸序列的各变体,所述变体含有一个或多个符合副球菌种菌株1534密码子利用表(参见表14)的取代,或SEQ ID NO157的1185-1610位核苷酸序列的片段,该片段编码具有1-脱氧木酮糖-5-磷酸合成酶活性的多肽。该实施方案还包括在严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针的核苷酸序列由SEQ ID NO157或其互补序列的1185-1610位的至少30个连续核苷酸构成,其中该多核苷酸编码具有1-脱氧木酮糖-5-磷酸合成酶活性的多肽。
优选地,该分离的多核苷酸由SEQ ID NO157的1185-1610位核苷酸构成。
本发明的另一实施方案是具有SEQ ID NO177的核苷酸序列的分离多核苷酸序列,SEQ ID NO177核苷酸序列的各变体,所述变体含有一个或多个符合副球菌种菌株1534密码子利用表(参见表14)的取代,或SEQ IDNO177核苷酸序列的片段,该片段编码具有选自下述活性的多肽乙酰-CoA乙酰基转移酶和乙酰乙酰-CoA还原酶。该实施方案还包括在如上定义的严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针的核苷酸序列由SEQ ID NO177或其互补序列的至少30个连续核苷酸构成,其中该多核苷酸编码具有选自乙酰-CoA乙酰基转移酶或乙酰乙酰-CoA还原酶活性的多肽。
该实施方案中,分离的多核苷酸序列还可包括SEQ ID NO177的1-1170位核苷酸序列,SEQ ID NO177核苷酸序列的各变体,所述变体含有一个或多个符合副球菌种菌株1534密码子利用表(参见表14)的取代,或SEQ ID NO177核苷酸序列的片段,该片段编码具有乙酰-CoA乙酰基转移酶活性的多肽。该实施方案还包括在严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针的核苷酸序列由SEQ ID NO177或其互补序列的1-1170位的至少30个连续核苷酸构成,其中该多核苷酸编码具有乙酰-CoA乙酰基转移酶活性的多肽。
优选地,该分离的多核苷酸序列由SEQ ID NO177的1-1170位核苷酸构成。
该实施方案中,分离的多核苷酸序列还可以选择地是SEQ ID NO177的1258-1980位核苷酸序列,SEQ ID NO177核苷酸序列的各变体,所述变体含有一个或多个符合副球菌种菌株1534密码子利用表(参见表14)的取代,或SEQ ID NO177核苷酸序列的片段,该片段编码具有乙酰乙酰-CoA还原酶活性的多肽。该实施方案还包括在严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针的核苷酸序列由SEQ ID NO177或其互补序列的1258-1980位的至少30个连续核苷酸构成,其中该多核苷酸编码具有乙酰乙酰-CoA还原酶活性的多肽。
优选地,该分离的多核苷酸序列由SEQ ID NO177的1258-1980位核苷酸构成。
在另一优选实施方案中,分离的多核苷酸序列具有选自SEQ ID NO42,SEQ ID NO157,SEQ ID NO177或其组合的核苷酸序列。本文的词组“或其组合”,当用于指核苷酸序列时,是指可对所提及序列的各种组合进行结合以形成该分离的多核苷酸序列。而且,在本发明中,可使用相同序列的多个拷贝,即多连体(concatamers)。同样,如同以下将要详细论述的,含有相同多核苷酸序列的质粒多拷贝可被转移到适当的宿主细胞中。
在本文中,“分离的”多核苷酸(如RNA,DNA或混合的多聚物)是实质上与其它细胞组分分离开的多核苷酸,而这些组分如核糖体,聚合酶,许多其它基因组序列和蛋白,在天然条件下是与天然序列或多肽相互混合的。该术语包括从天然存在的环境中被分离出来的多核苷酸,还包括重组或克隆的DNA分离物,以及化学合成的类似物或由异源系统生物合成的类似物。
术语“核酸序列”指从5′到3′末端方向阅读的、单链或双链的脱氧核糖核苷酸或核糖核苷酸碱基多聚物。其包括染色体DNA,自我复制的质粒,感染性DNA或RNA多聚体,以及具有一级结构功能的DNA或RNA。
“表达调控序列”指核苷酸调控序列的排列,其指导着可操作性连接的核酸的转录。这种表达调控序列的实例为“启动子”。启动子包括在转录起始位点附近的必要的核酸序列。启动子还可任选地含有末端(distal)增强子或阻遏元件,增强子或阻遏元件可定位在转录起始位点的几千个碱基对之外。“组成型”启动子是指在多数环境条件和发育条件下都能够激活的启动子。而“诱导型”启动子是指在环境因素或发育因素的调节下被激活的启动子。术语“可操纵性连接/可操作相连”指在核酸表达调控序列(如启动子或转录因子结合位点的排列)和第二个核酸序列之间的功能性连接,其中表达调控序列指导着相应的第二个序列核酸的转录。
如果一段多核苷酸序列来自异源物种,或者虽然来自相同物种,但已被修饰成与原来不同的形式,那么这段多核苷酸则与某种有机体或第二个多核苷酸序列是“异源的”。例如,启动子与异源编码序列可操纵连接是指编码序列的来源物种不同于启动子的来源物种,或者,来自相同物种,但是编码序列已被修饰成与天然存在的等位变体不同的形式。
当同时发生转基因的表达和外源基因的抑制(如通过反义作用,或有意义抑制(sense suppression))时,本领域技术人员将会意识到插入的多核苷酸序列不必要完全相同于其所来源于的基因序列,但是必须是“基本上相同的”。
当插入的多核苷酸序列被转录以及翻译以生成功能性多肽时,本领域技术人员会认识到由于密码子简并性,大量的多核苷酸序列都可以编码相同的多肽。这些变体都具体包括在本发明的范围之内。另外,本发明还具体包括彼此基本上相同(如下所述进行测定)的序列,并且其编码的多肽是野生型多肽的突变体,或仍维持多肽的功能(如,该多肽氨基酸的保守取代而形成的)。另外,变体可以编码如下所述的显性阴性突变(negative mutant)。
按如下所述,在两个核酸序列或两个多肽序列是按照最大一致性排列时,如果两序列中核苷酸序列或氨基酸残基是分别相同的,可以说这两个核酸序列或这两个多肽是“相同的”。术语“相同”或“同一性”百分比,当用于描述两个或多个核酸或多肽序列时,指当在比较窗口下对比并排列其最大一致性时(按照下述序列对比算法之一进行测定,或通过人工排列和目测进行测定),两个或多个序列或亚序列是相同的,或者具有特定百分比的相同氨基酸残基或核苷酸。当序列同一性的百分比用于蛋白或多肽时,就认为不相同的残基位置通常因保守氨基酸取代而不同,其中氨基酸残基是被其它类似化学属性(如电荷或疏水性)的氨基酸取代,从而不改变该分子的功能。当序列以保守取代的形式而各不相同时,序列同一性百分比可被上调,以校正取代的保守性性质。进行这种调整的方法是本领域技术人员所公知的。通常包括以部分而不是全部错配的方式对保守取代打分(scoring),从而提高序列同一性的百分比。这样,例如,当评定相同的氨基酸为1分时,非保守取代的得分为0,而保守取代的得分在0到1之间。保守取代的得分可根据例如Meyers & Miller,Computer Applic.Biol.Sci.411-17(1988)的算法进行计算,如同在PC/GENE(Intelligenetics,MountainView,Calif.,USA)程序中所执行的。
短语“实质上相同(substantially identical)”,当用于形容两个核酸或多肽时,指当在比较窗口下对比并排列最大一致性时(按照下述序列对比算法之一进行测定,或通过人工排列和目测进行测定),序列间或亚序列间具有至少60%、优选80%,最优选90-95%的核苷酸或氨基酸残基同一性。该定义还指一种序列,该序列的互补序列能够与检测序列杂交。
进行序列对比时,通常一个序列作为对照序列,将检测序列与其进行对比。当使用序列对比算法时,检测和对照序列都被输入计算机,如果必要的话,还可指定各个并列(coordinate)的亚序列,并制定序列算法程序的各个参数。可使用默认的程序参数,或指定可选参数。然后,序列对比算法可在程序参数基础上,计算检测序列相对于对照序列的序列同一性百分比。
本文所用的“对比窗口”,包括选自以下任何数目的连续位置片段参照体系20-600,通常约50-约200,更通常约100-约150,其中序列可以与相同数目的连续位置的对照序列在这两个序列被最优化排列之后,进行对比。排列序列用于对比的方法是本领域公知的。用于对比的序列优化排列可通过如Smith和Waterman,Adv.Appl.Math.2482(1981)的局部同源性算法进行,如Needleman和Wunsch,J.Nat’l,Acad.Sci.USA 852444(1988)的同源性排列算法,这些算法(Wisconsin Genetics Software Package,GeneticsComputer Group,575 Science Dr.,Madison,Wis.中的GAP,BESTFIT,FASTA,和TFASTA)的计算机化运行,或人工排列和目测(visualinspection)。
一种有用的算法实例是PILEUP。PILEUP从一组相关的序列中,使用渐进性成对排列的方法,产生出多个序列排列,以显示其相关性和序列同一性百分比。它还绘出树形图或树状图(dendogram),以标明用于形成排列的聚类(clustering)关系。PILEUP使用简化的Feng和Doolittle,J.Mol.Evol.35351-360(1987)渐进性排列方法。所用的该方法类似于Higgins和Sharp,CABIOS 5151-153(1989)所描述的方法。该程序可以序列对比(align)达300个序列,每个序列的最大长度为5000个核苷酸或氨基酸。多元排列方法是以两个最近似序列的配对排列开始,形成了两个排列序列的组(cluster)。然后该组与下一个最相关的序列或排列序列的组相排列。两组序列通过两个单个序列的成对排列的简单延伸而排列。一系列渐进性配对排列之后就获得了最后的排列。通过指定用于序列比较区域的具体序列和其氨基酸或核苷酸序列匹配物(coordinate),并指定程序的参数,就可运行该程序。例如,对照序列可与其它的检测序列相比较,以测定序列同一性关系的百分比,使用以下参数默认的gap weight(3.00),默认的gap length weight(0.10)和weighted end gaps。
另一种适用于测定序列同一性百分比以及序列相似性的算法的实例为BLAST算法[Altschul等,J.Mol.Biol.215403-410(1990)]。进行BLAST分析的软件可通过National Center for Biotechnology Information(http//www.ncbi.nlm.nih.gov/)获得。该算法涉及首先通过在查询序列中鉴定出W长度的短序列(words),当该序列与数据库序列中相同长度的序列对比时,能够匹配或符合某个以位置进行衡量(positive-valued)的阈值T,以此鉴定出高分的序列对(HSP)。T指临近序列(neighborhood word)的分数阈值(Altschul等,同上)。这些起始性临近序列点作为种子(seeds)用于起始检索,以找到将之包含其中的更长的HSP。序列点(hit)在每个序列的两个方向上均延伸,以使累积的排列分数能够增加。当下述情况下,停止序列点在两个方向上的延伸累积的排列分数从其获得的最大值处下降了X数量;由于一个或多个负分数残基加入排列后逐渐积累,使得累积的分数变为0或负数,或者任一序列已到达末尾。该BLAST算法参数W,T和X决定着排列的灵敏度和速度。使用BLAST程序的默认参数序列长度(word length)(W)为11,BLOSUM62分数基质(参见Henikoff和Henikoff,Proc.Natl.Acad.SciUSA 8910915(1989))排列(B)为50,预期值(E)为10,M=5,N=-4,以及两条链的对比。
BLAST算法还可对两条序列之间的相似性进行统计学分析[例如,参见Karlin和Altschul,Proc.Nat’l.Acad.Sci.USA 905873-5787(1993)]。BLAST算法提供的相似性测定方法是概率的最小和(P(N)),其表明两个核苷酸或氨基酸序列之间偶然发生匹配的机率。例如,如果检测核酸与对照核酸之间的对比中,最小总和概率(smallest sum probability)小于约0.2,更有选小于约0.01,最优选小于约0.001,则认为该核酸与对照核酸相似。
“保守修饰的变体”可用于描述氨基酸或核酸序列。当用于特定核酸序列时,保守修饰的变体指编码相同或基本相同氨基酸序列的核酸,或不编码氨基酸序列的核酸,基本相同的序列。由于遗传密码的简并性,许多功能相同的核酸密码子可编码任何指定的蛋白。例如,密码子GCA,GCC,GCG和GCU都编码丙氨酸。这样,在每个丙氨酸被某个密码子具体指定的各位置,该密码子都可被改变为任何相应所述的密码子,而不会改变被编码的多肽。这样的核酸变体是“沉默变体”(silent variations),其为一种保守修饰的变体。本文中编码多肽的每个核酸序列还描述该核酸的各个可能沉默变体。本领域技术人员公知核酸中的每个密码子(除了AUG,其通常为蛋氨酸的唯一密码子)均可被修饰,以形成功能相同的分子。同样,编码多肽的核酸的每个沉默突变均已隐含在每个被描述的序列中。
当描述氨基酸序列时,本领域技术人员公知核酸中单个的取代,缺失或插入,或肽,多肽或蛋白序列的取代,其改变了编码序列中的单个氨基酸或小百分率氨基酸(即,小于20%,如15%,10%,5%,4%,3%,2%或1%),当这种改变形成了类似化学属性氨基酸的氨基酸取代时,则认为该核酸序列为“保守取代变体”。提供功能类似氨基酸的保守取代表是本领域公知的。
下述六组均含有彼此为保守取代的氨基酸丙氨酸(A),丝氨酸(S),苏氨酸(T);天冬氨酸(D),谷氨酸(E);天冬酰胺(N),谷氨酰胺(Q);精氨酸(R),赖氨酸(K);异亮氨酸(I),亮氨酸(L),蛋氨酸(M),缬氨酸(V);和苯丙氨酸(F),酪氨酸(Y),色氨酸(W)。(参见,如Creighton,Proteins(1984))。
指示两个核酸序列或多肽基本上相同的标识为第一个核酸编码的多肽,与抗第二个核酸所编码多肽的抗体之间具有免疫交叉反应。这样,例如,当一个多肽与第二个多肽之间的区别仅仅在于保守取代时,这两个多肽通常实质上(substantially)相同。指示两个核酸序列实质上相同的标识为这两个分子或其互补序列彼此之间能够在如下所述的严谨条件下杂交。
短语“与……特异性杂交”指当核酸序列存在于复杂混合物(如总细胞或DNA或RNA文库)中时,在严谨杂交条件下,某分子仅仅与该特定的核酸序列结合,配对(duplexing)或杂交。
短语“严谨杂交条件”指在该条件下,探针能够与其靶序列而非其它序列杂交,所述靶序列通常存在于核酸序列的复杂混合物中。严谨杂交条件是序列依赖性的,并在不同的环境条件下有所不同。长序列在较高的温度下才会特异杂交。核酸杂交的详细指南可参见Tijssen,Techniques inBiochemistry and Molecular Biology-Hybridization with Nucleic Probes,“Overviews of Principles of Hybridization and the Strategy of Nucleic AcidAssays”(1993)。通常,高度严谨条件选择为在指定的离子强度和pH下,比特定序列的热融点(Tm)低约5-10℃。低严谨条件通常选择为Tm下约15-30℃。Tm是指这样的温度在该温度下(在指定离子强度,pH,和核酸浓度下),有50%互补于靶序列的探针以平衡状态与该靶序列杂交(当靶序列过量存在时,在Tm下,50%的探针被结合,该状态维持平衡)。严谨条件指这样的条件在pH7.0-8.3下,盐浓度低于约1.0M钠离子,通常约0.01-1.0M钠离子浓度(或其它盐),对于短探针(如10-50个核苷酸)而言,温度至少约为30℃,而长探针(如大于50个核苷酸)至少约为60℃。严谨条件还可用加入去稳定剂如甲酰胺而获得。为获得选择性或特定杂交,阳性信号应至少是杂交背景的2倍,优选10倍。
如果它们所编码的肽实质上是相同的,在严谨条件下彼此并不杂交的核酸实质上仍然是相同的。例如当使用该遗传密码的最大密码子简并性形成核酸拷贝时,这种情况会发生。在这种情形下,核酸通常在中等程度的严谨条件下杂交。
在本发明中,含有本发明核酸的基因组DNA或cDNA可使用本发明公开的核酸序列,在严谨条件下,用标准Southern印迹鉴定。为此目的,适于这种杂交的严谨条件包括在37℃,在40%甲酰胺,1M NaCl,1%十二烷基硫酸钠(SDS)的缓冲液中杂交,并在至少约50℃,通常为约55℃-约60℃,用0.2X SSC至少进行20分钟的洗涤一次,或等价条件进行。阳性杂交至少是背景的2倍。本领域普通技术人员公知其它可选的杂交条件和洗涤条件也可用于提供类似的严格度条件。
进一步指示两多核苷酸实质上相同的标识为如果用一对寡核苷酸引物扩增对照序列,该对照序列在严谨杂交条件下用作探针,用于从cDNA或基因组文库中分离检测序列,或鉴定检测序列,如在northern或Southern印迹中。
本发明还包括如上定义的表达载体。该表达载体包括一个或多个上述每种多核苷酸序列的拷贝。本发明的表达载体可含有本文所定义的任一多核苷酸序列,如SEQ ID NO42,或SEQ ID NO42的下述残基2622-3644,3641-4690,4687-5853,5834-6970,6970-7887,7880-8878,以及SEQ ID NO157的残基59-292,295-1158或1185-1610,SEQ ID NO177的残基1-1170或1258-1980。该表达载体可含有本文鉴定的多核苷酸序列的各组合,如,SEQ ID NO42,SEQ ID NO157和SEQ ID NO177。
该表达载体中的多核苷酸还可如上定义,任选地与表达调控序列可操作相连并如实施例中进行扩增。
本发明还包括以下表达载体pBBR-K-mev-op16-1,pBBR-K-mev-op16-2,pDS-mvaA,pDS-idi,pDS-hcs,pDS-mvk,pDS-pmk,pDS-mvd,pDS-His-mvaA,pDS-His-idi,pDS-His-hcs,pDS-His-mvk,pDS-His-pmk,pDS-His-mvd,pBBR-K-Zea4,pBBR-K-Zea4-up,pBBR-K-Zea4-down,pBBR-K-PcrtE-crtE-3,pBBR-tK-PcrtE-mvaA,pBBR-tK-PcrtE-idi,pBBR-tK-PcrtE-hcs,pBBR-tK-PcrtE-mvk,pBBR-tK-PcrtE-pmk,pBBR-tK-PcrtE-mvd,pBBR-K-PcrtE-mvaA-crtE-3,pDS-His-phaA,pBBR-K-PcrtE-crtW,pBBR-K-PcrtE-crtWZ,pBBR-K-PcrtE-crtZW及其组合。这些表达载体在以下的实施例中定义更为详细。而且,本发明还包括含有本文所定义任一序列的表达载体,该表达载体被用于在适当的宿主细胞内表达类异戊二烯化合物,如类胡萝卜素,优选玉米黄质。本文中,短语“表达载体”是携带编码本文所述多核苷酸序列的DNA序列,并能够介导该序列表达的可复制载体。
在本文中,术语“可复制”指在该载体已经导入的、指定类型的宿主细胞中,该载体能够复制。目的多核苷酸序列的直接上游可以提供一段编码信号肽的序列,该肽的存在可以确保携带有载体的宿主细胞表达该编码多肽的分泌。该信号序列可以是与被选择多核苷酸序列天然相关连,或来自另外的来源。
该载体可以是任何可方便地进行重组DNA操作的载体,载体的选择通常依赖于该载体将要导入的宿主细胞。这样,该载体可以是自主复制载体,即,作为染色体外实体存在的载体,其复制独立于染色体的复制;这种载体的例子可包括质粒,噬菌体,粘粒或极微染色体。或者,该载体可以是这样的当其被导入宿主细胞时,其可以整合到宿主染色体组中,并随着该被整合的染色体一起复制。适当的载体如实施例所示。本发明的表达载体可以携带以下定义的本发明任一DNA序列,以及用于表达本发明以下定义的任何多肽。
本发明还包括含有本文所述的一个或多个多核苷酸序列和/或一个或多个表达载体的培养细胞。本文中,“培养细胞”包括任何能够在指定条件下生长并表达一个或多个本发明多核苷酸编码多肽的细胞。优选地,该培养细胞为酵母,真菌,细菌或藻类。更优选地,该培养细胞为副球菌,黄杆菌属(Flavobacterium),土壤杆菌属(Agrobacterium),产碱杆菌属(Alcaligenes),欧文氏杆菌属(Erwinia),大肠杆菌(E.coli)或枯草杆菌(B.subtilis)。甚至更优选地,该细胞为副球菌,如R-1506,R-1512,R1534或R114。本发明还包括本文所鉴定的、能够表达本文所公开多肽的任何细胞的后代。在本发明中,如果使用实施例2所述条件,某个细胞的AFLP DNA指纹与假定亲代细胞的无区别,则认为该细胞即为其后代。
这样,本发明的培养细胞可包括,例如SEQ ID NO42或SEQ ID NO42的下述残基2622-3644,3641-4690,4687-5853,5834-6970,6970-7887,7880-8878,以及SEQ ID NO157的残基59-292,295-1158或1185-1610,SEQ ID NO177的残基1-1170或1258-1980。这些序列可以单独地,或作为其它表达载体的一部分,被转移到细胞中。这些序列还可任选地与表达调控序列可操作相连。该培养细胞还可含有本文所鉴定多核苷酸序列的各个组合,如SEQ ID NO42,SEQ ID NO157和SEQ ID NO177。
本发明的培养细胞还可进一步含有编码一个或多个类胡萝卜素生物合成途径酶的多核苷酸。例如,本发明的培养细胞可含有一个或多个SEQ IDNO180,182和184的拷贝,这些拷贝可以单独或与本文所定义的任一多核苷酸序列一起包含在本发明培养细胞中。这样,本发明公开的多核苷酸序列可以单独地,或与其它能够提高类异戊二烯化合物靶物质(如象玉米黄质或虾青素的类胡萝卜素)产量的另一多核苷酸序列一起,被转移到培养细胞中。在这方面,本发明包括任何编码如类胡萝卜素生物合成相关多肽的多核苷酸的应用,所述多肽如GGPP合成酶,β-胡萝卜素-β4-加氧酶(酮酶),和/或β-胡萝卜素羟化酶。另外,还可将编码类胡萝卜素生物合成相关多肽的多核苷酸组合与一个或多个本文鉴定的多核苷酸在相同或不同的表达载体中联合使用。这种构建体可被转移到本发明的培养细胞中以形成能够表达目的类异戊二烯物质的细胞。
例如,本发明的培养细胞可含有一个或多个下述表达载体pBBR-K-mev-op16-1,pBBR-K-mev-op16-2,pDS-mvaA,pDS-idi,pDS-hcs,pDS-mvk,pDS-pmk,pDS-mvd,pDS-His-mvaA,pDS-His-idi,pDS-His-hcs,pDS-His-mvk,pDS-His-pmk,pDS-His-mvd,pBBR-K-Zea4,pBBR-K-Zea4-up,pBBR-K-Zea4-down,pBBR-K-PcrtE-crtE-3,pBBR-tK-PcrtE-mvaA,pBBR-tK-PcrtE-idi,pBBR-tK-PcrtE-hcs,pBBR-tK-PcrtE-mvk,pBBR-tK-PcrtE-pmk,pBBR-tK-PcrtE-mvd,pBBR-K-PcrtE-mvaA-crtE-3,pDS-His-phaA,pBBR-K-PcrtE-crtW,pBBR-K-PcrtE-crtWZ,pBBR-K-PcrtE-crtZW及其组合。
本发明的另一实施方案为生产类胡萝卜素的方法。该方法中,在能够表达如上定义多核苷酸序列所编码的多肽的条件下,培养如上定义的培养细胞。能够表达多肽的培养条件如下述实施例所述,但如需要可进行修改,以适应特定目的需求。然后从细胞或者如果分泌,从细胞培养基中分离类胡萝卜素。
本发明中,“类胡萝卜素”包括下述化合物八氢番茄红素,番茄红素,β-胡萝卜素,玉米黄质,角黄素,虾青素,福寿草黄素(adonixanthin),隐黄素,海胆酮和福寿草红素(adonirubin)及其组合。优选的类胡萝卜素为玉米黄质。
本发明的另一实施方案为制备产类胡萝卜素细胞的方法。该方法包括(a)将编码甲羟戊酸途径酶的多核苷酸序列导入细胞,所述酶在该细胞中能够表达;以及(b)选择出含有步骤(a)多核苷酸序列的细胞,并且该细胞所产生类胡萝卜素的水平为该细胞导入多核苷酸序列之前的1.1-1000倍。
本文中,短语“甲羟戊酸途径酶”指IPP生物合成的甲羟戊酸途径中相关的各种酶,并由atoB或phaA,hcs,mvaA,mvk,pmk和mvd基因编码。就本发明的目的而言,如果使用实施例1所示的任一活性试验能够检测到某种酶,则该酶“在细胞内表达”。检测类胡萝卜素产量的各方法是本领域公知的。实施例1,11和12分别提供了鉴定玉米黄质,番茄红素和虾青素的存在的常规试验方法。类似地,其它类胡萝卜素检测试验可用于检测细胞或培养基中如八氢番茄红素,角黄素,福寿草黄素,隐黄素,海胆酮和福寿草红素的存在。
这样,该方法可用于下述示范性类胡萝卜素的测定八氢番茄红素,番茄红素,β-胡萝卜素,玉米黄质,角黄素,虾青素,福寿草黄素(adonixanthin),隐黄素,海胆酮和福寿草红素(adonirubin)及其组合。该方法中,优选的类胡萝卜素为玉米黄质。
该方法包括制备一种细胞,该细胞生成类胡萝卜素的水平约为该细胞导入多核苷酸序列之前时的1.1-1000倍,优选约1.5-500倍,例如约100倍,或至少10倍。
该方法中,细胞生成类胡萝卜素的量约为1mg/L-10g/L。优选细胞的类胡萝卜素产量为100mg/L-约9g/L,例如,约500mg/L-8g/L,或约1g/L-5g/L。
该方法中,细胞可选自酵母,真菌,细菌或藻类。优选地,该细胞为副球菌(Paracoccus),黄杆菌属(Flavobacterium),土壤杆菌属(Agrobacterium),产碱杆菌属(Alcaligenes),欧文氏杆菌属(Erwinia),大肠杆菌(E.coli)或枯草杆菌(B.subtilis)。更优选地,该细胞为副球菌。
该方法中,细胞可以是突变细胞。本文中“突变细胞”是指任何含有非天然多核苷酸序列、或含有已被改变(如通过重排或缺失或取代1-100个,优选20-50个,更优选低于10个核苷酸)为不同于天然形式的多核苷酸序列的细胞。这种非天然序列可通过随机诱变,化学诱变,UV辐射等等获得。优选地,该突变导致一种或多种甲羟戊酸途径的基因表达量提高,从而提高类胡萝卜素如玉米黄质的产量。制备,筛选和鉴定这种突变细胞的方法是本领域公知的,并如以下的实施例所示。这种突变体的例子为R114或R1534。优选地,该突变细胞为R114。
该方法中,多核苷酸序列为SEQ ID NO42或SEQ ID NO42的下述残基2622-3644,3641-4690,4687-5853,5834-6970,6970-7887,7880-8878,以及SEQ ID NO157的残基59-292,295-1158或1185-1610,SEQ ID NO177的残基1-1170或1258-1980。这些序列可以单独地,或作为其它表达载体的一部分使用。这些序列还可任选地与表达调控序列可操作相连。在该方法中,还可以使用本文所鉴定多核苷酸序列的各个组合,如SEQ ID NO42,SEQ ID NO157和SEQ ID NO177。
可被选择用于该方法的表达载体包括pBBR-K-mev-op16-1,pBBR-K-mev-op16-2,pDS-mvaA,pDS-idi,pDS-hcs,pDS-mvk,pDS-pmk,pDS-mvd,pDS-His-mvaA,pDS-His-idi,pDS-His-hcs,pDS-His-mvk,pDS-His-pmk,pDS-His-mvd,pBBR-K-Zea4,pBBR-K-Zea4-up,pBBR-K-Zea4-down,pBBR-K-PcrtE-crtE-3,pBBR-tK-PcrtE-mvaA,pBBR-tK-PcrtE-idi,pBBR-tK-PcrtE-hcs,pBBR-tK-PcrtE-mvk,pBBR-tK-PcrtE-pmk,pBBR-tK-PcrtE-mvd,pBBR-K-PcrtE-mvaA-crtE-3,pDS-His-phaA,pBBR-K-PcrtE-crtW,pBBR-K-PcrtE-crtWZ,pBBR-K-PcrtE-crtZW及其组合。
该方法中,可使用任何常规方法将多核苷酸序列导入细胞。将多核苷酸序列导入细胞的适当方法包括转化,转导,转染,脂转染(lipofection),电穿孔[参见如Shigekawa和Dower,Biotechniques 6742-751(1988)],接合[参见如Koehler和Thorne,Journal of Bacteriology 1695771-5278(1987)]和基因枪法(biolistics)。
接合在转移多核苷酸序列中的应用,例如以表达载体的形式将多核苷酸转移到受体细菌中的应用通常是有效的,也是熟知的方法(如US5,985,623)。根据细菌的菌株,更通常使用感受态细胞与纯化DNA进行转化。
公知的电穿孔技术(体外和体内)是通过向定位于处理区域附近的电极施加短暂高压脉冲而进行的。在电极间所形成的电场会在细胞膜上产生瞬时形成的小孔,通过该小孔,外源物质就可进入细胞。已知的电穿孔应用中,该电场含有在约100μs的时期中,1000V/cm数量级的单一方形波脉冲。这样的脉冲可通过例如BTX Division of Genetronics,Inc制造的ElectorSquare PoratorT820的已知应用来产生。
基因枪法是运用微弹轰击技术,将多核苷酸运送到靶细胞的系统。通过加速将多核苷酸运送到靶细胞的方法的说明性实施方案为BiolisticsParticle Delivery System,其可用于推进DNA或细胞包覆的颗粒通过一个屏幕,如不锈钢或Nytex屏幕,到达覆盖有培养细胞的过滤器表面。该屏幕会使颗粒散开,这样颗粒不会过于集中地到达培养细胞。据认为居于离子轰击仪和待轰击细胞细胞之间的屏幕会减少粒子凝集的大小,可以通过减少由于过大微弹攻击受体细胞而造成的损失,从而提高转化频率。
为进行轰击,悬浮液中的细胞优选被浓缩到过滤器或固体培养基上。或者,可将其它靶细胞处理到固体培养基上。待轰击的细胞被固定到微弹阻停板下面适当的距离处。如果需要,还可在加速装置和待轰击细胞之间固定一个或多个屏幕。利用这些已知的技术,可以获得高于1000或更多数目的瞬时表达标记基因的细胞焦点。焦点中,轰击后48小时表达外源基因产物的细胞数目通常为1-10,平均为1-3。
在轰击转化中,可将轰击前培养条件和轰击参数最优化,以产生最大数目的稳定转化体。该项技术中轰击的物理和生物学参数都很重要。物理因素是那些与处置多核苷酸/-微弹沉淀相关的因素,或者是影响大或小轰击弹飞行和速度的因素。生物学因素包括在轰击之前和之后立即操作细胞的相关所有步骤,帮助减轻与轰击相关创伤的靶细胞渗透压的调整,还包括转化DNA的性质,如线性DNA或完整的超螺旋质粒。
相应地,可以考虑小范围地调整各种轰击参数以达到条件的最优化。技术人员可能特别期望调整物理参数,如间隙距(gap distance),飞行距离,组织距离(tissue distance)和氦压。技术人员还可以通过修饰影响受体细胞生理状态的条件从而影响转化和整合的效率,将损伤简化因子(traumareduction factors)(TRFs)最小化。例如,可以调整渗透状态,组织水合作用和受体细胞的传代培养阶段或细胞周期来优选转化。参照本文,本领域技术人员将公知其它常规的调整操作。
微粒介导的转化方法是本领域技术人员公知的。例如,US 5,015,580(将其引入本文作为参考)描述了使用这种技术进行的大豆转化。
本发明的另一实施方案是对细菌进行改造使其生产类异戊二烯化合物的方法。这样的细菌是按如下制备(a)在能够表达类异戊二烯的条件下,在培养基中培养亲代细菌,并从该培养基中筛选出突变细菌,该细菌生产的类异戊二烯水平为亲代细菌的约1.1-1000倍;(b)将含有SEQ ID NO42多核苷酸序列的表达载体导入突变细菌,该多核苷酸序列与表达调控序列可操作相连;以及(c)筛选出含有表达载体并且生产类异戊二烯的水平比步骤(a)中的突变体高出至少约1.1倍的细菌。
在该实施方案中,类异戊二烯化合物指以异戊烯二磷酸(IPP)单元的下述通式为结构基础的化合物。
这种化合物包括半萜,单萜,倍半萜,双萜,三萜(如植物甾醇,植物雌激素(phytoestrogens),植物昆虫脱皮激素,雌激素,植物雌激素(phytoestrogens)),四萜(类胡萝卜素)和多萜。优选地,类异戊二烯为类胡萝卜素,例如上述鉴定的类胡萝卜素之一,特别是玉米黄质。
细菌可以是使用本发明公开方法能够生成类异戊二烯化合物的任何细菌。优选地,该细菌为副球菌(Paracoccus),黄杆菌属(Flavobacterium),土壤杆菌属,产碱杆菌属(Alcaligenes),欧文氏菌属(Erwinia),大肠杆菌或枯草杆菌。甚至更优选地,该细胞为副球菌。优选地,亲代细菌为R-1506或R1512,突变细菌为R1534或R114,优选为R114。
在优化生产类异戊二烯的条件下,在培养基中培养细菌。培养基的选择和培养条件是本领域公知的。实施例1,11和12中所示分析试验提供测定培养基中某种类胡萝卜素的存在的示范性试验方法。通过优化培养条件以及测定靶类异戊二烯的产量,可以实现符合本文所述特定产量参数的突变株的培养和筛选。以这种方法,可以筛选出类异戊二烯生产水平比亲代细菌高约1.1-1000倍的突变细菌。优选地,该突变细菌生产的类异戊二烯比亲代细菌高约1.5-500倍,例如,比亲代细菌高至少约100倍或至少约10倍。然后培养该细菌,用于后续的步骤。
筛选出能够以期望的水平生产类异戊二烯的突变细菌后,使用前述的或实施例中所述的任一方法将表达载体导入该细菌。本文所定义的任一表达载体均可导入该突变细胞。优选地,该表达载体含有SEQ ID NO42。一旦将表达载体导入突变细菌后,筛选出类异戊二烯产量比未转化突变体高出至少约1.1倍,例如约5-20倍的稳定转化体。然后将该选出的转化体在适于类异戊二烯生成的条件下培养,接着从细胞或培养基中分离出类异戊二烯。
该方法的进一步步骤为向突变细菌中引入突变,该突变能够提高细菌的类异戊二烯化合物的产量。该突变可选自下述的至少一种失活聚羟基烷羧酸酯(polyhydroxyalkanoate)(PHA)途径,提高乙酰-CoA乙酰基转移酶的表达,提高FPP合成酶的表达,提高类胡萝卜素生物合成途径中酶的表达,提高转化异戊烯二磷酸(IPP)为二甲基丙烯二磷酸(dimethylallyldiphosphate)(DMAPP)的酶的表达。
失活聚羟基烷羧酸酯(polyhydroxyalkanoate)(PHA)途径可通过选择出不表达phaB(SEQ ID NO177的1258-1980位核苷酸)编码多肽的突变细菌而完成,或者使用SEQ ID NO177或其片段进行同源重组,通过破坏野生型phaB基因的表达而完成。
该方法中,提高乙酰-CoA乙酰基转移酶的表达可通过下述完成将含有SEQ ID NO175或SEQ ID NO177的1-1170位核苷酸所示多核苷酸序列的载体导入突变细菌,所述多核苷酸序列与表达调控序列可操作相连。该方法中,提高FPP合成酶的表达可通过下述完成将含有SEQ ID NO157的295-1158位核苷酸所示多核苷酸序列的载体导入突变细菌,所述多核苷酸序列与表达调控序列可操作相连。该方法中,提高类胡萝卜素基因的表达可通过下述完成将载体导入突变细菌,所述载体含有编码类胡萝卜素生物合成途径中一种或多种酶的多核苷酸序列,如选自下述的多核苷酸序列SEQ ID NO180,182和184,所述多核苷酸序列与表达调控序列可操作相连。
该方法中,优选类异戊二烯化合物为异戊烯二磷酸(IPP)。还优选该类异戊二烯化合物为类胡萝卜素,例如八氢番茄红素,番茄红素,β-胡萝卜素,玉米黄质,角黄素,虾青素,福寿草黄素(adonixanthin),隐黄素,海胆酮和福寿草红素(adonirubin)及其组合。
本发明的另一实施方案为副球菌属(Paracoccus)的微生物,该微生物具有下述特点(a)使用GeneCompar v2.0软件,缺口罚分(gap penalty)为0%条件下,利用同源性计算获得的相似度基质,与SEQ ID NO12的序列相似性大于97%;(b)在81.5℃,使用DNA:DNA杂交,与R-1512,R1534,R114或R-1506的相似性大于70%;(c)基因组DNA的G+C含量与R114,R-1512,R1534和R-1506的染色体组DNA的G+C含量相比,区别小于1%;以及(d)使用实施例2的AFLP方法,平均DNA指纹与菌株R-1512,R1534,R114和R-1506具有cluster约58%的相似性,附带条件为该微生物不属于副球菌种(MBIC3966)。
测定上述每一特征的方法在实施例2中充分描述,可以预期当使用这些方法时,很容易地检测出符合上述标准的微生物。优选本发明的微生物具有上述的每项特征(即a-d)。但是,a-d任一特征的组合也属于本发明的范围,只要该组合能够提供足够的信息可以确凿地从分类学角度描述与R114,R-1512,R1534和R-1506属于相同种的微生物,除副球菌种之外(MBIC3966)。
本发明的另一实施例是副球菌属的微生物,该微生物具有下述特征(a)含有至少约75%的细胞膜总脂肪酸的18:1w7c;(b)不能使用福寿草醇(adonitol),i-赤藓糖醇,龙胆二糖,β-甲基葡糖苷,D-山梨糖醇,木糖醇和奎尼酸作为碳源用于生长;和(c)使用L-天冬酰胺和L-天冬氨酸作为碳源用于生长,附带条件为该微生物不属于副球菌种(MBIC3966)。
实施例2也详细描述了测定每项这些特征的方法,可以预期当使用这些方法时,可以容易地检测出符合上述标准的微生物。优选本发明的微生物具有上述的每项特征(即a-c)。但是,a-c任一特征的组合也属于本发明的范围,只要该组合能够提供足够的信息可以确凿地从分类学角度描述与R114,R-1512,R1534和R-1506属于相同种的微生物,除副球菌种之外(MBIC3966)。
本发明的另一实施方案是副球菌属的微生物,该微生物具有下述特征(a)能够在40℃生长;(b)能够在含有8%NaCl的培养基中生长;(c)能够在pH9.1的培养基中生长;以及(d)黄色-橙色集落色素形成,附带条件为该微生物不属于副球菌种(MBIC3966)。
实施例2也详细描述了测定上述每项特征的方法,可以预期当使用这些方法时,可以容易地检测出符合上述标准的微生物。优选本发明的微生物具有上述的每项特征(即a-d)。但是,a-d任一特征的组合也属于本发明的范围,只要该组合能够提供足够的信息可以确凿地从分类学角度描述与R114,R-1512,R1534和R-1506属于相同种的微生物,除副球菌种之外(MBIC3966)。
本发明的微生物还可以使用上述11项特征的组合进行定义,只要该组合能够提供足够的信息可以确凿地从分类学角度描述与R114,R-1512,R1534和R-1506属于相同种的微生物,除副球菌种之外(MBIC3966)。
综上所述,本发明提供(1)含有选自下述氨基酸序列的分离多肽(a)SEQ ID NO43的1-340残基所示的氨基酸序列,特别是相应于SEQID NO43的68-97位的氨基酸序列;(b)SEQ ID NO45的1-349残基所示的氨基酸序列,特别是相应于SEQID NO45的1-30位的氨基酸序列;(c)SEQ ID NO47的1-388残基所示的氨基酸序列,特别是相应于SEQID NO47的269-298位的氨基酸序列;(d)SEQ ID NO49的1-378残基所示的氨基酸序列,特别是相应于SEQID NO49的109-138位的氨基酸序列;(e)SEQ ID NO51的1-305残基所示的氨基酸序列,特别是相应于SEQID NO51的198-227位的氨基酸序列;(f)SEQ ID NO53的1-332残基所示的氨基酸序列,特别是相应于SEQID NO53的81-110位的氨基酸序列;(g)选自SEQ ID NO43,45,47,49,51或53的氨基酸序列的片段,其中所述片段具有至少30个连续的氨基酸残基;(h)选自SEQ ID NO43,45,47,49,51或53的多肽片段的氨基酸序列,该片段具有羟基甲基戊二酸单酰-CoA还原酶(HMG-CoA还原酶),异戊烯二磷酸异构酶,羟基甲基戊二酸单酰-CoA合成酶(HMG-CoA合成酶),甲羟戊酸激酶,磷酸甲羟戊酸激酶,或二磷酸甲羟戊酸脱羧酶的活性;(i)在严谨条件下能够与杂交探针杂交的多核苷酸所编码的多肽氨基酸序列,所述杂交探针含有至少SEQ ID NO42或SEQ ID NO42互补序列的30个连续多核苷酸,其中所述多肽具有HMG-CoA还原酶,异戊烯二磷酸异构酶,HMG-CoA合成酶,甲羟戊酸激酶,磷酸甲羟戊酸激酶,或二磷酸甲羟戊酸脱羧酶的活性;以及(j)SEQ ID NO43,45,47,49,51或53经保守修饰而形成的各变体。
(2)含有选自下述氨基酸序列的分离多肽(a)SEQ ID NO159的1-287残基所示氨基酸序列;(b)SEQ ID NO159的至少30个连续氨基酸残基;(c)SEQ ID NO159的片段的氨基酸序列,该片段具有法呢基二磷酸合成酶(FPP合成酶)的活性;(d)在严谨条件下能够与杂交探针杂交的多核苷酸所编码的多肽氨基酸序列,所述杂交探针含有至少SEQ ID NO157或其互补序列的295-1158位的30个连续多核苷酸,其中所述多肽具有FPP合成酶的活性;以及(e)SEQ ID NO159经保守修饰而形成的变体。
(3)含有选自下述氨基酸序列的分离多肽(a)SEQ ID NO160的1-142残基所示氨基酸序列;(b)SEQ ID NO160的至少30个连续氨基酸残基;(c)SEQ ID NO160的片段的氨基酸序列,该片段具有1-脱氧木酮糖-5-磷酸合成酶(DXPS)的活性;(d)在严谨条件下能够与杂交探针杂交的多核苷酸所编码的多肽氨基酸序列,所述杂交探针含有至少SEQ ID NO157或其互补序列的1185-1610位的30个连续多核苷酸,其中所述多肽具有DXPS的活性;以及(e)SEQ ID NO160经保守修饰而形成的各变体。
(4)含有选自下述氨基酸序列的分离多肽(a)SEQ ID NO178的1-390位残基所示氨基酸序列;(b)SEQ ID NO178的至少30个连续氨基酸残基;(c)SEQ ID NO178的多肽片段的氨基酸序列,该片段具有乙酰-CoA乙酰基转移酶的活性;(d)在严谨条件下能够与杂交探针杂交的多核苷酸所编码的多肽氨基酸序列,所述杂交探针含有至少SEQ ID NO177或其互补序列的1-1170位的30个连续多核苷酸,其中所述多肽具有乙酰-CoA乙酰基转移酶的活性;以及
(e)SEQ ID NO178经保守修饰而形成的变体。
(5)含有选自下述氨基酸序列的分离多肽(a)SEQ ID NO179的1-240位残基所示氨基酸序列;(b)SEQ ID NO179的至少30个连续氨基酸残基;(c)SEQ ID NO179的多肽片段的氨基酸序列,该片段具有乙酰乙酰-CoA还原酶的活性;(d)在严谨条件下能够与杂交探针杂交的多核苷酸所编码的多肽氨基酸序列,所述杂交探针含有至少SEQ ID NO177或其互补序列的1258-1980位的30个连续多核苷酸,其中所述多肽具有乙酰乙酰-CoA还原酶的活性;以及(e)SEQ ID NO179经保守修饰而形成的各变体。
(6)含有选自下述核苷酸序列的分离多核苷酸序列SEQ ID NO42,SEQ ID NO42变体的核苷酸序列,所述变体含有一个或多个符合副球菌种菌株R1534密码子利用表(参见表14)的取代,SEQ ID NO42的片段,该片段编码具有选自下述活性的多肽羟基甲基戊二酸单酰-CoA还原酶(HMG-CoA还原酶),异戊烯二磷酸异构酶,羟基甲基戊二酸单酰-CoA合成酶(HMG-CoA合成酶),甲羟戊酸激酶,磷酸甲羟戊酸激酶和二磷酸甲羟戊酸脱羧酶,在严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针由SEQ ID NO42或其互补序列的至少30个连续核苷酸构成,该多核苷酸编码具有选自下述活性的多肽HMG-CoA还原酶,异戊烯二磷酸异构酶,HMG-CoA合成酶,甲羟戊酸激酶,磷酸甲羟戊酸激酶和二磷酸甲羟戊酸脱羧酶;特别是(a)分离的多核苷酸序列,其含有的多核苷酸序列选自SEQ ID NO42的2622-3644位核苷酸,SEQ ID NO42的片段,其编码具有HMG-CoA还原酶活性的多肽,以及在严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针由SEQ ID NO42或其互补序列2622-3644位残基的至少30个连续核苷酸构成,所述多核苷酸编码具有HMG-CoA还原酶活性的多肽,尤其是SEQ ID NO42的2622-3644位核苷酸构成的序列;(b)分离的多核苷酸序列,其含有的多核苷酸序列选自SEQ ID NO42的3641-4690位核苷酸,其各种变体,所述变体含有一个或多个符合副球菌种菌株R1534密码子利用表(参见表14)的取代,SEQ ID NO42的片段,该片段编码具有异戊烯二磷酸异构酶活性的多肽,以及在严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针由SEQ ID NO42或其互补序列3641-4690位残基的至少30个连续核苷酸构成,所述多核苷酸编码具有异戊烯二磷酸异构酶活性的多肽,尤其是SEQ ID NO42的3641-4690位核苷酸构成的多核苷酸序列;(c)分离的多核苷酸序列,其含有的多核苷酸序列选自SEQ ID NO42的4687-5853位核苷酸,其各种变体,所述变体含有一个或多个符合副球菌种菌株R1534密码子利用表(参见表14)的取代,SEQ ID NO42的片段,该片段编码具有HMG-CoA合成酶活性的多肽,以及在严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针由SEQ ID NO42或其互补序列4687-5853位残基的至少30个连续核苷酸构成,所述多核苷酸编码具有HMG-CoA合成酶活性的多肽,尤其是SEQ ID NO42的3641-4690位核苷酸构成的序列;(d)分离的多核苷酸序列,其含有的多核苷酸序列选自SEQ ID NO42的5834-6970位核苷酸,其各种变体,所述变体含有一个或多个符合副球菌种菌株R1534密码子利用表(参见表14)的取代,SEQ ID NO42的片段,该片段编码具有甲羟戊酸激酶活性的多肽,以及在严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针由SEQ ID NO42或其互补序列5834-6970位残基的至少30个连续核苷酸构成,所述多核苷酸编码具有甲羟戊酸激酶活性的多肽,尤其是SEQ ID NO42的3641-4690位核苷酸构成的多核苷酸序列;(e)分离的多核苷酸序列,其含有的多核苷酸序列选自SEQ ID NO42的6970-7887位核苷酸,其各种变体,所述变体含有一个或多个符合副球菌种菌株R1534密码子利用表(参见表14)的取代,SEQ ID NO42的片段,该片段编码具有磷酸甲羟戊酸激酶活性的多肽,以及在严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针由SEQ ID NO42或其互补序列6970-7887位残基的至少30个连续核苷酸构成,所述多核苷酸编码具有磷酸甲羟戊酸激酶活性的多肽,尤其是SEQ ID NO42的3641-4690位核苷酸构成的多核苷酸序列;(f)分离的多核苷酸序列,其含有的多核苷酸序列选自SEQ ID NO42的7880-8878位核苷酸,其各种变体,所述变体含有一个或多个符合副球菌种菌株R1534密码子利用表(参见表14)的取代,SEQ ID NO42的片段,该片段编码具有二磷酸甲羟戊酸脱羧酶活性的多肽,以及在严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针由SEQ ID NO42或其互补序列7880-8878位残基的至少30个连续核苷酸构成,所述多核苷酸编码具有二磷酸甲羟戊酸脱羧酶活性的多肽,尤其是SEQ ID NO42的3641-4690位核苷酸构成的多核苷酸序列;(7)含有选自下述多核苷酸序列的分离多核苷酸序列SEQ ID NO157的核苷酸序列,SEQ ID NO157各变体,所述变体含有一个或多个符合副球菌种菌株R1534密码子利用表(参见表14)的取代,SEQ ID NO157的片段,该片段编码具有选自下述活性的多肽法呢基焦磷酸(FPP)合成酶活性,1-脱氧-D-木酮糖-5-磷酸合成酶活性,或编码具有XseB活性的多肽,以及在严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针由SEQ IDNO157或其互补序列的至少30个连续核苷酸构成,该多核苷酸编码具有选自下述活性的多肽FPP合成酶活性,1-脱氧-D-木酮糖-5-磷酸合成酶活性,以及XseB活性,特别是(a)分离的多核苷酸序列,其含有的多核苷酸序列选自SEQ ID NO157的59-292位核苷酸序列,SEQ ID NO157各变体,所述变体含有一个或多个符合副球菌种菌株R1534密码子利用表(参见表14)的取代,SEQ IDNO157的59-292位核苷酸序列的片段,该片段编码具有XseB功能的多肽,以及在严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针由SEQID NO157或其互补序列59-292位残基的至少30个连续核苷酸构成,所述多核苷酸编码具有XseB功能的多肽,尤其是SEQ ID NO157的59-292位核苷酸构成的序列;(b)分离的多核苷酸序列,其含有的多核苷酸序列选自SEQ ID NO157的295-1158位核苷酸序列,SEQ ID NO157的295-1158位核苷酸序列的变体,所述变体含有一个或多个符合副球菌种菌株R1534密码子利用表(参见表14)的取代,SEQ ID NO157的295-1158位核苷酸序列的片段,该片段编码具有FPP合成酶活性的多肽,以及在严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针由SEQ ID NO157或其互补序列295-1158位残基的至少30个连续核苷酸构成,所述多核苷酸编码具有FPP合成酶活性多肽,尤其是SEQ ID NO157的295-1158位核苷酸构成的多核苷酸序列;
(c)分离的多核苷酸序列,其含有的多核苷酸序列选自SEQ ID NO157的1185-1610位核苷酸序列,SEQ ID NO157的1185-1610位核苷酸序列的各变体,所述变体含有一个或多个符合副球菌种菌株R1534密码子利用表(参见表14)的取代,SEQ ID NO157的1185-1610位核苷酸序列的片段,该片段编码具有1-脱氧木酮糖-5-磷酸合成酶活性的多肽,以及在严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针由SEQ ID NO157或其互补序列1185-1610位残基的至少30个连续核苷酸构成,所述多核苷酸编码具有1-脱氧木酮糖-5-磷酸合成酶活性多肽,尤其是SEQ ID NO157的1185-1610位核苷酸构成的序列;(8)含有选自下述多核苷酸序列的分离多核苷酸序列SEQ ID NO177的核苷酸序列,SEQ ID NO177各变体,所述变体含有一个或多个符合副球菌种菌株R1534密码子利用表(参见表14)的取代,SEQ ID NO177的片段,该片段编码具有选自下述活性的多肽乙酰-CoA乙酰基转移酶活性和乙酰乙酰-CoA还原酶活性,以及在严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针由SEQ ID NO177或其互补序列的至少30个连续核苷酸构成,该多核苷酸编码具有选自下述活性的多肽乙酰-CoA乙酰基转移酶和乙酰乙酰-CoA还原酶,特别是(a)分离的多核苷酸序列,其含有的多核苷酸序列选自SEQ ID NO177的1-1170位核苷酸序列,SEQ ID NO177各变体,所述变体含有一个或多个符合副球菌种菌株R1534密码子利用表(参见表14)的取代,SEQ IDNO177的片段,该片段编码具有乙酰-CoA乙酰基转移酶活性的多肽,以及在严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针由SEQ IDNO177或其互补序列1-1170位残基的至少30个连续核苷酸构成,所述多核苷酸编码具有乙酰-CoA乙酰基转移酶活性的多肽,尤其是SEQ ID NO177的1-1170位核苷酸构成的序列;(b)分离的多核苷酸序列,其含有的多核苷酸序列选自SEQ ID NO177的1258-1980位核苷酸序列,SEQ ID NO177各变体,所述变体含有一个或多个符合副球菌种菌株R1534密码子利用表(参见表14)的取代,SEQ IDNO177的片段,该片段编码具有乙酰乙酰-CoA还原酶活性的多肽,以及在严谨条件下能够与杂交探针杂交的多核苷酸序列,所述探针由SEQ IDNO177或其互补序列1258-1980位残基的至少30个连续核苷酸构成,所述多核苷酸编码具有乙酰乙酰-CoA还原酶活性的多肽,尤其是SEQ ID NO177的1258-1980位核苷酸构成的序列;(9)含有选自下述核苷酸序列的分离多核苷酸序列SEQ ID NO42,SEQ ID NO157,SEQ ID NO177及其组合;(10)含有(6)(a)-(6)(f),(7)(a)-(7)(c),(8)(a),(8)(b)或(9)任一多核苷酸序列的表达载体,特别是这样的表达载体其中多核苷酸序列与表达调控序列可操作相连,例如进一步含有编码类胡萝卜素生物合成途径中酶的多核苷酸序列的表达载体,更特别是其中所含多核苷酸序列选自下述的表达载体SEQ ID NO180,SEQ ID NO182,SEQ ID NO184及其组合,该多核苷酸序列与表达调控序列可操作相连;(11)选自下述的表达载体pBBR-K-mev-op16-1,pBBR-K-mev-op16-2,pDS-mvaA,pDS-idi,pDS-hcs,pDS-myk,pDS-pmk,pDS-mvd,pDS-His-mvaA,pDS-His-idi,pDS-His-hcs,pDS-His-mvk,pDS-His-pmk,pDS-His-mvd,pBBR-K-Zea4,pBBR-K-Zea4-up,pBBR-K-Zea4-down,pBBR-K-PcrtE-crtE-3,pBBR-tK-PcrtE-mvaA,pBBR-tK-PcrtE-idi,pBBR-tK-PcrtE-hcs,pBBR-tK-PcrtE-mvk,pBBR-tK-PcrtE-pmk,pBBR-tK-PcrtE-mvd,pBBR-K-PcrtE-mvaA-crtE-3,pDS-His-phaA,pBBR-K-PcrtE-crtW,pBBR-K-PcrtE-crtWZ,pBBR-K-PcrtE-crtZW及其组合,特别是(a)选自pBBR-K-mev-op16-1或pBBR-K-mev-op16-2的表达载体,(b)选自pBBR-K-Zea4,pBBR-K-Zea4-up或pBBR-K-Zea4-down的表达载体,(c)选自pBBR-K-PcrtE-crtE-3,pBBR-tK-PcrtE-mvaA,pBBR-tK-PcrtE-idi,pBBR-tK-PcrtE-hcs,pBBR-tK-PcrtE-mvk,pBBR-tK-PcrtE-pmk,pBBR-tK-PcrtE-mvd或其组合的表达载体,(d)表达载体pBBR-K-PcrtE-mvaA-crtE-3,(e)表达载体pDS-His-phaA,或(f)选自pBBR-K-PcrtE-crtW,pBBR-K-PcrtE-crtWZ或pBBR-K-PcrtE-crtZW的表达载体;(12)含有(6)(a)-(f),(7)(a)-(c),(8)(a),(8)(b)或(9)任一多核苷酸序列的培养细胞,或含有(10)或(11)表达载体的培养细胞,或该细胞的后代,其中该细胞表达由所述多核苷酸编码的多肽,特别是进一步具有选自下述特征的细胞(a)进一步含有编码类胡萝卜素生物合成途径中酶的多核苷酸序列,特别是这样的培养细胞该细胞中编码类胡萝卜素生物合成途径酶的多核苷酸序列是选自SEQ ID NO180,SEQ ID NO182,SEQ ID NO184,或者是该细胞的后代,其中该细胞表达由所述多核苷酸序列编码的多肽,以及(b)选自酵母,真菌,细菌和藻类,特别是选自下述的细菌副球菌,黄杆菌属(Flavobacterium),土壤杆菌属(Agrobacterium),产碱杆菌属(Alcaligenes),欧文氏杆菌属(Erwinia),大肠杆菌(E.coli)或枯草杆菌(B.subtilis),更具体是副球菌,尤其是选自R-1506,R-1512,R1534或R114的副球菌。
(13)生产类胡萝卜素的方法,包括在能够表达多核苷酸序列编码多肽的条件下培养(12)细胞,并从细胞或该细胞的培养基中分离出类胡萝卜素;(14)制备产类胡萝卜素细胞的方法,包括(a)向细胞中导入编码甲羟戊酸途径中酶的多核苷酸序列,所述酶在该细胞中表达;以及(b)选择出含有步骤(a)多核苷酸序列的细胞,并且该细胞所产生类胡萝卜素的水平为该细胞导入多核苷酸序列之前的1.1-1000倍,特别是以下述特点为特征的方法(i)筛选步骤包括选择出含有步骤(a)多核苷酸序列的细胞,且该细胞所产生类胡萝卜素的水平约为该细胞导入多核苷酸序列之前的1.5-500倍,特别是约100倍,或至少约10倍;(ii)该细胞的类胡萝卜素产量约为1mg/L-约10g/L。
(ii)该细胞选自酵母,真菌,细菌和藻类,特别是选自下述的细菌副球菌,黄杆菌属(Flavobacterium),土壤杆菌属(Agrobacterium),产碱杆菌属(Alcaligenes),欧文氏杆菌属(Erwinia),大肠杆菌(E.coli)或枯草杆菌(B.subtilis),更具体是副球菌;(iii)步骤(a)的细胞为突变细胞,特别是选自R114和R1534的细胞,特别是这样的突变细胞其类胡萝卜素化合物产量约为其非突变亲代的1.1-1000倍,特别是约1.5-500倍,更特别是至少约高100倍,或至少高10倍;
(v)选自(6)(a)-(f),(7)(a)-(c),(8)(a),(8)(b)或(9)的多核苷酸序列,特别是与表达调控序列可操作相连的多核苷酸序列;(vi)表达载体(10)或(11)的多核苷酸序列;(vii)导入步骤选自转化,转导,转染,脂转染,电穿孔,接合或基因枪法。
(viii)类胡萝卜素选自八氢番茄红素,番茄红素,β-胡萝卜素,玉米黄质,角黄素,虾青素,福寿草黄素(adonixanthin),隐黄素,海胆酮和福寿草红素(adonirubin)及其组合;特别是类胡萝卜素为玉米黄质。
(15)改造细菌使其生成类异戊二烯化合物的方法,包括(a)在能够表达类异戊二烯化合物的条件下,在培养基中培养亲代细菌,并从该培养基中筛选出突变细菌,所述突变细菌类异戊二烯化合物产量比亲代细菌高约1.1-1000倍;(b)向该突变细菌中导入含有SEQ ID NO42所示多核苷酸序列的表达载体,所述多核苷酸序列与表达调控序列可操作相连;以及(c)筛选出含有该表达载体的细菌,并且所述细菌的类异戊二烯化合物产量比步骤(a)的突变株至少高1.1倍,尤其是(i)进一步向突变细菌导入突变的方法,更特别是所述突变产生下述至少一种作用的方法使聚羟基链羧基酯(PHA)途径失活,提高乙酰-CoA乙酰基转移酶的表达,提高法呢基焦磷酸(FPP)合成酶的表达,提高类胡萝卜素途径酶的表达,提高转化异戊烯二磷酸(IPP)为二甲基烯丙基焦磷酸(DMAPP)的酶的表达,最尤其是使聚羟基链羧基酯(PHA)途径失活的方法,包括筛选出突变细菌,该细菌不表达phaB(SEQ ID NO177的1258-1980位核苷酸)编码的多肽,或者该方法包括通过使用SEQ ID NO177或其片段进行同源重组,从而破坏野生型phaB基因的表达,或者一种提高乙酰-CoA乙酰基转移酶的表达的方法,包括向突变细菌中导入含有SEQ ID NO175所示核苷酸序列或SEQ ID NO177 1-1170位核苷酸的载体,所述核苷酸序列与表达调控序列可操作相连,或一种提高FPP合成酶的表达的方法,包括向突变细菌中导入含有SEQID NO157的295-1158位核苷酸所示多核苷酸序列的载体,所述核苷酸序列与表达调控序列可操作相连,或一种提高类胡萝卜素途径酶的表达的方法,包括向突变细菌中导入载体,所述载体含有选自下述的多核苷酸序列SEQ ID NO180,182和184,且该多核苷酸与表达调控序列可操作相连,(a)一种方法,其中类异戊二烯为异戊烯二磷酸(IPP)。
(b)一种方法,其中类异戊二烯为类胡萝卜素,特别是类胡萝卜素选自下述的方法八氢番茄红素,番茄红素,β-胡萝卜素,玉米黄质,角黄素,虾青素,福寿草黄素(adonixanthin),隐黄素,海胆酮和福寿草红素(adonirubin)及其组合;(c)一种方法,其中亲代细菌为副球菌,特别是R1512或R-1506,或R1534或R114,特别是突变株为R114;(16)副球菌属微生物,其具有下述特点(i)使用利用GeneCompar v2.0软件,缺口罚分(gap penalty)为0%条件下,利用同源性计算获得的相似度基质(similarity matrix),与SEQ ID NO12的序列相似性大于97%;在81.5℃,使用DNA:DNA杂交,与R-1512,R1534,R114或R-1506的同源大于70%;基因组DNA的G+C含量与R114,R-1512,R1534和R-1506的基因组DNA的相比,区别小于1%;以及使用实施例2的AFLP方法,平均DNA指纹与菌株R-1512,R1534,R114和R-1506具有约58%的相似性,附带条件为该微生物不属于副球菌种(MBIC3966);(ii)含有至少约75%的细胞膜总脂肪酸的18:1w7c;能够抑制使用福草糖醇(adonitol),i-赤藓糖醇,龙胆二糖,β-甲基葡糖苷,D-山梨糖醇,木糖醇和奎尼酸作为碳源用于生长;和能够使用L-天冬酰胺和L-天冬氨酸作为碳源用于生长,附带条件为该微生物不属于副球菌种(MBIC3966),或(iii)能够在40℃生长;能够在含有8%NaCl的培养基中生长;能够在pH9.1的培养基中生长;以及黄色-橙色集落色素形成,附带条件为该微生物不属于副球菌种(MBIC3966)。
下述实施例用于进一步说明本发明各个方面。这些实施例仅为说明目的,而非限制本发明的范围。
实施例1分析方法和生化方法(a)类胡萝卜素的分析样品制备首先制备二甲基亚砜(DMSO)和四氢呋喃的1∶1溶剂混合物。加入丁化羟基甲苯(butylated hydroxytoluene)(BHT,0.5g/l溶剂混合物)稳定溶剂混合物。向装有0.4ml细菌培养物的一次性15ml聚丙烯离心管中加入4毫升稳定了的DMSO/THF混合物(最终稀释因子为1/11)。将这些离心管盖严,每个离心管均用Vortex混合仪混合10秒钟。然后将该样品放入Brinkmann Vibramix振荡器内振荡20分钟。室温下,4000rpm离心4分钟,将各份澄清的黄色/橙色上清液分别转移到棕色玻璃瓶中,用于高效液相色谱(HPLC)。
HPLC发展一种反向HPLC方法,用于同时测定虾青素,玉米黄质,角黄素,β-胡萝卜素和番茄红素。该方法能够将玉米黄质的各主要顺式异构体分离开。使用带有恒温自动取样器和二极管阵列检测仪的Agilent 1100 HPLC系统进行色谱。该方法的各参数如下柱 YMC类胡萝卜素C30柱,微粒大小为5微米250*4.6mm I.D.钢YMC,Part No.CT99S052546WT)Guard柱Pelliguard LC-18 cartridge,20mm(SUPELCO,Part No.59654)移动相 甲醇(MeOH)/甲基特丁基醚(TBME)梯度
进行时间28分钟;典型柱压力初始90巴;流速1.0ml/min;检测450nm紫外光;加入体积10μl;柱温15℃试剂甲醇和TBME为HPLC级,分别来自EM Science和J.T.Baker。DMSO(Omnisolve)购自EM Science。THF(HPLC溶剂)来自Burdick和Jackson。
计算使用外标准(由Hoffmann-La Roche,Basel,Switzerland提供),用二级校准(two level calibration)进行定量计算。计算基于峰面积。
选择性该方法的选择性依据相关类胡萝卜素对照化合物标准溶液的加入而得到检验。靶化合物(全反式类胡萝卜素(all-trans-carotenoids))被彻底分离,并未显示干涉。一些次要的顺式异构体可以共洗脱,尽管这些潜在的干涉异构体非常少量,可以在常规分析中忽略不计。组合物的滞留时间如表1所列。
表1 类胡萝卜素的HPLC滞留时间
直线性 将25毫克全反式玉米黄质溶于50ml DMSO/THF混合物(玉米黄质终浓度为500μg/ml)。制备稀释系列(玉米黄质终浓度为250,100,50,10,5,1和0.1μg/ml),并用上述的HPLC方法进行分析。发现线性范围在0.1μg/ml-250μg/ml。相关系数为0.9998。
检测的极限值该方法对玉米黄质进行检测的检测下限被测定为60μg/l。更高的注射体积以及优化整合参数可以将检测极限减低到约5μg/l。
重现性全顺式玉米黄质的滞留时间非常稳定(相对标准偏差(RSD),0.2%)。根据对相同培养样品进行的10次重复性分析,峰面积重现性测定为全反式玉米黄质0.17%RSD,隐黄素1.0%。
(b)制备粗提物以及酶试验方法制备粗提物用1ml抽提缓冲液(视被试验的酶而定——组分与下述的各个酶学试验方法而具体说明)重悬浮洗涤的细胞沉淀,制备副球菌和大肠杆菌粗提物。将细胞悬浮液置于2ml塑料瓶中,并使用Mini Bead Beater 8(BiospecProducts,Bartlesville OK USA)玻璃珠振荡,破碎沉淀。然后在4℃,使用培养基振荡器进行破裂。在4℃下,21000xg离心该破裂的沉淀制备溶液20分钟,使细胞碎片沉淀,上清液直接用于酶试验。
蛋白检测使用Bio-Rad Protein Assay Reagent(Bio-Rad,Hercules,CA,USA),通过Bradford[Anal.Biochem.72248-2254(1976)]的方法测定粗提物中的蛋白浓度。使用牛血清白蛋白作为对照蛋白,用于构建标准曲线。
乙酰-CoA乙酰基转移酶试验粗提物在pH8.0的150mM EPPS(N-[2-羟乙基]piperizine-N’-[3-propanesulfonic acid])缓冲液中制备。根据Slater等人[J.Bacteriol.1801979-1987(1998)]的描述,在硫解作用方向上进行试验。该试验在304nm下,利用分光光度计测量乙酰乙酰-CoA的消失。反应混合物含有150mM EPPS缓冲液(pH8.0),50mM MgCl2,100μM CoA,40μM乙酰乙酰-CoA和粗提物。反应在30℃进行,通过加入粗提物起始反应。使用SpectraMAX Plus板式读数器(Molecular Devices Corp.,Sunnyvale,CA,USA)和石英微量滴定板(也可使用任何标准分光光度计),在304nm下监测乙酰乙酰-CoA的消失。使用乙酰乙酰-CoA(1单位活性=1μmol被消耗的乙酰乙酰-CoA/分钟)制备的标准曲线,计算活性(表达为U/mg蛋白)。乙酰-CoA乙酰基转移酶活性的检测下限为0.006U/mg。
HMG-CoA合成酶试验根据Honda等人[Hepatology 27154-159(1998)]的方法试验HMG-CoA合成酶。该试验中,从乙酰-CoA和乙酰乙酰-CoA形成的HMG-CoA通过由HPLC分离反应产物和底物直接进行测定。粗提物在50mM Tris-HCl缓冲液中(pH8.0)制备。反应混合物(0.1ml)含有50mMTris-HCl缓冲液(pH8.0),0.1mM EDTA,20mM MgCl2,0.1mM乙酰乙酰-CoA,0.8mM乙酰-CoA和粗提物。加入粗提物之前,在30℃预温育反应混合物2分钟。在30℃反应5分钟后,加入0.2ml 200mM四丁基磷酸铵(TBAP,溶解在甲醇-水(3∶2,终pH为5.5)中,并含有0.2mM丙酰-CoA作为内部回收标准)终止反应。然后在4℃,21000xg离心混合物3分钟,并随即保存在冰上,直到通过反相离子对HPLC进行分析。使用Nova-Pak C18柱(3.9×150mm,Waters Corporation,Milford,MA,USA),从乙酰-CoA和乙酰乙酰-CoA中分离出HMG-CoA和丙酰-CoA。注入体积为20μl,移动相为溶于甲醇-水(1∶1,终pH为5.5)的50mM TBAP,流速为1.0ml/min。通过在254nm下的吸收值来检测HMG-CoA和丙酰-CoA。该反应中形成的HMG-CoA与使用可信HMG-CoA制备的标准曲线进行对比。活性定义为U/mg蛋白。1单位活性=生成1nmol HMG-CoA/分钟。HMG-CoA合成酶的检测下限约为1U/mg。
HMG-CoA还原酶试验粗提物在含有50mM KCl,1mM EDTA和蛋白酶抑制剂混合物(protease inhibitor cocktail)(Sigma Chemical Co.,St.Louis,MO,USA,catalog#P-2714)的25mM磷酸钾缓冲液(pH7.2)中制备。根据Takahashi等人[J.Bacteriol.1811256-1263(1999)]的方法进行试验。该方法在340nm下,利用分光光度计测量HMG-CoA依赖性NADPH的氧化。反应混合物含有25mM磷酸钾缓冲液(pH7.2),50mM KCl,1mM EDTA,5mM二硫苏糖醇,0.3mM NADPH,0.3mM R,S-HMG-CoA和粗提物。反应在30℃下进行,加入HMG-CoA起始反应。使用SpectraMAX Plus板式读数器(Molecular Devices Corp.,Sunnyvale,CA,USA)和石英微量滴定板(也可使用任何标准分光光度计),在340nm下监测HMG-CoA依赖性NADPH氧化。使用NADPH制备的标准曲线,计算酶活(表达为U/mg蛋白)(1单位活性=氧化NADPH 1μmol/分钟)。HMG-CoA还原酶活性的检测下限为0.03U/mg。甲羟戊酸激酶,磷酸甲羟戊酸激酶和甲羟戊酸二磷酸脱羧酶试验制备底物,用于甲羟戊酸激酶、磷酸甲羟戊酸激酶和甲羟戊酸二磷酸脱羧酶的试验方法已在Popják[Methods Enzymol.15393-425(1969)]中详细描述。所有的试验中,1单位酶活均定义为生成的1μmol产物/分钟。除了分光光度计和放射性色谱试验,还可使用其他的方法,如使用HPLC来分离反应底物和产物。甲羟戊酸激酶、磷酸甲羟戊酸激酶和甲羟戊酸二磷酸脱羧酶的检测下限通常约为0.001U/mg蛋白。
IPP异构酶试验粗提物被制备到50mM Tris-HCl缓冲液(pH7.5)中。使用Spurgeon等人[Arch.Biochme.Biophys.230445-545(1984)]的方法进行试验。该反析是基于IPP和DMAPP之间酸不稳定性的差异。反应混合物(终体积0.1ml)含有50mM Tris-HCl缓冲液(pH7.5),2mM二硫苏糖醇,5mM MgCl2,20μM[1-14C]-IPP和粗提物。在30℃下反应15分钟,加入0.3ml浓缩的HCl∶甲醇(4∶1)混合物终止反应,并额外在37℃下孵育20min。加入己烷(0.9ml),混合试管(使用涡旋混合器,10秒,4次)。离心(21000xg,5分钟)后,将0.6ml的己烷层转移到闪烁瓶内,加入闪烁液,计数放射性。活性表达为U/mg蛋白。1单位活性=1pmol被掺入到酸不稳定性产物的[1-14C]-IPP/分钟)。IPP异构酶活性的检测下限为1U/mg。
FPP合成酶粗提物被制备到50mM Tris-HCl缓冲液(pH8.0)中。FPP合成酶试验方法类似于上述的IPP异构酶试验,是基于IPP和FPP之间酸不稳定性的差异[Spurgeon等,同上]。反应混合物(终体积0.1ml)含有50mM Tris-HCl缓冲液(pH8.0),2mM二硫苏糖醇,5mM MgCl2,20μM[1-14C]-IPP,25μMGPP(gernayl pyrophosphate)和粗提物。在30℃下反应15分钟,加入0.3ml浓缩的HCl∶甲醇(4∶1)混合物终止反应,并额外在37℃下孵育20min。加入己烷(0.9ml),混合试管(使用涡旋混合器,10秒,4次)。离心(21000xg,5分钟)后,将0.6ml的己烷层转移到闪烁管内,加入闪烁液,计数放射性。酶活性单位和检测下限,与上述IPP异构酶的定义相同。当较高的IPP异构酶活性干扰了FPP合成酶活性的测定时,可将粗提物在5mM碘乙酰胺的存在下预温育5分钟,以抑制IPP异构酶的活性。
GGPP合成酶试验粗提物被制备到含有2mM二硫苏糖醇的50mMTris-HCl(pH8.0)中。根据Kuzuguchi等人[J.Biol.Chem.2745888-5894(1999)]的方法对GGPP合成酶进行分析。该试验是基于与上述FPP合成酶相同的原理。反应混合物(终体积0.1ml)含有50mM Tris-HCl缓冲液(pH8.0),2mM二硫苏糖醇,5mM MgCl2,20μM[1-14C]-IPP,25μM FPP和粗提物。所有的反应条件和随后用于闪烁计数的样品处理均与上述FPP合成酶的相同。还可使用上面所述的碘乙酰胺处理抽提物,以抑制IPP异构酶的活性。酶活单位和检测下限与上述IPP异构酶的定义相同。
乙酰乙酰-CoA还原酶试验粗提物被制备到含有50mM KCl和5mM二硫苏糖醇的50mM Tris-HCl(pH8.0)中。根据Chohan和Copeland[Appl.Environ.Microbiol.642859-2863(1998)]的方法进行乙酰乙酰-CoA还原酶试验。该试验在340nm下,利用分光光度计测定乙酰乙酰-CoA依赖性的NADPH氧化。反应混合物(1ml)含有50mM Tris-HCl缓冲液(pH8.5),15mM MgCl2,250μM NADPH,和100μm乙酰乙酰-CoA。反应在30℃,在石英比色杯中进行,加入乙酰乙酰-CoA起始反应。使用NADPH制备的标准曲线,计算酶活(表达为U/mg蛋白)(1单位活性=1μmol被氧化的NADPH/分钟)。乙酰乙酰-CoA还原酶活性的检测下限约为0.01U/mg。
实施例2从分类学角度将黄杆菌属菌种重新分类为副球菌该实施例说明了将早期命名为黄杆菌属的菌株R1512(ATCC 21588)从分类学角度重新分类为副球菌种菌株R-1512(ATCC 21588)。全面的基因组和生化/生理分析由Belgian Coordinated Collections ofMicroorganisms/Laboratorium voor Microbiologie,Universiteit Gent(BCCMTM/LMG)进行,使用目前公知的本领域常规技术作为细菌分类科学标准。除副球菌种菌株R-1512之外,其他几种属于副球菌属的细菌也包括在该研究中(归纳在表2中)。
表2.用于分类学研究的细菌
菌株R1534和R114是通过经典诱变技术由菌株R-1512突变而形成,利用改进的玉米黄质产量而将其筛选出来。初级筛选是选出形成色彩强度最大的集落。次级筛选是根据实施例1所述HPLC方法在液体培养基中进行。菌株R-1506是从提供菌株R-1512的相同起始筛选中从环境微生物中独立分离而获得。菌株MBIC3024,MBIC3966,MBIC4017,MBIC4020根据其16S rDNA基因的核苷酸序列(DNA序列存储在公共EMBL数据库中,参见表5),均被鉴定为副球菌属的成员。最近,paracoccus marcusii DSM11574T和Paracoccus carotinifaciens E-396T被认定为属于产类胡萝卜素细菌类型[Harker等,Int.J.Syst.Bacteriol.48543-548(1998);Tsubokura等,Int.J.Syst.Bacteriol.49277-282(1999)]。Paracoccus solventivorans DSM 6637T作为“对照”菌株,该菌株属于副球菌属,但是与所用的其它细菌亲缘关系较远。
初步的试验产生了下述结果。本文所述的各种方法能够清楚地鉴定有机体相似性的分类学关系或相关程度。Van Damme等人MicrobiologicalReviews 60407-438(1996)和Jassen等人Microbiology 1421881-1893(1996)中详细地描述并比较了这些方法及其在描述细菌门类上的应用。
(1)对菌株R1534和R114细胞膜的脂肪酸分析显示这两个菌株高度相似,说明了这两个菌株与Paracoccus denitrificans和Rhodobacter capsulatus的分类学相关度。
(2)细胞蛋白的单向凝胶电泳显示在R1534和R114之间存在极高的相似度(即,种内水平相关度),但是图谱均未表明这些菌株属于R.capsulatus或P.denitrificans。
(3)菌株R1534与R.capsulatus LMG2962T和P.denitrificans LMG4218T之间的DNA:DNA杂交证实了菌株R1534既不是R.capsulatus也不是P.denitrificans。
(4)菌株R1534和R114的16SrDNA基因测序显示这些有机体属于副球菌属,但是它们代表了新的种。最高程度的序列相似性是副球菌种菌株MBIC3966,MBIC4020和MBIC3024的16SrDNA基因。
(5)使用扩增片段长度多态性(AFLPTM)进行的菌株R1534和R114 DNA指纹图谱显示这两个菌株基因组DNA之间综合相似性极高,表明种内相关度(即AFLPTM可以在相同种内的两个成员间有所差异)。
下述部分是本发明对副球菌种菌株R-1512(及其突变株衍生的R1534和R114)所进行的大量分类学研究的结果和结论。
16S rDNA测序和系统发育研究表2所述的细菌培养在LMG培养基185((TSA)BBL 11758中,如果需要的话,可在培养基中补充1.5%的DifcoBacto琼脂)。根据Niemann等人[J.Appl.Microbiol.82477-484(1997)]的方法,制备基因组DNA。利用聚合酶链式反应(PCR),使用表3所示引物,从菌株R-1512,R1534和R-1506的基因组DNA中扩增编码16SrDNA的基因。
表3.PCR扩增副球菌种菌株R-1512,R1534和R-1506中编码16SrDNA的DNA所用引物
aF,正向引物;R,反向引物。正向引物16F27(又称PA)用于R1534和R-1506,而正向引物16F38(又称ARI C/T)用于菌株R-1512和R114。反向引物16R1522(又称PH)用于所有菌株。
b杂交位置指大肠杆菌16SrDNA基因序列编号。
使用Qiaquick PCR Purification Kit(Qiagen GmbH,Hilden,Germany)纯化PCR扩增的DNA。使用Applied Biosystems,Inc.337测序仪和操作手册(Perkin-Elmer,Applied Biosystems Division,Foster City,CA,USA)所述方法,使用“ABI PRISMTM Big DyeTM Terminator Cycle Sequencing ReadyReaction Kit(用AmpliTaq_DNA聚合酶Fs)”,进行完整的测序。DNA测序所用引物如表4所示。
表4.用于测序副球菌种菌株R-1512,R1534,R114和R-1506中编码16SrDNA基因的PCR扩增片段的引物
aF,正向引物;R,反向引物。
b杂交位置指大肠杆菌16S rDNA基因序列编号。
使用5个正向引物和3个反向引物制备部分重叠的序列,确保得到高度可信的组合序列数据。将共有序列(来自R-1512,R1534,R114和R-1506)包含在小核糖体亚基序列的排列之后(所述小核糖体亚基序列由国际核苷酸序列文库EMBL收集),使用AutoAssembler程序(Perkin-Elmer,AppliedBiosystems Division,Foster City,CA,USA)进行序列组合。使用GeneComparTM软件包(v2.0,Applied Maths B.V.B.A.,Kortrijk,Belgium)进行系统发育分析。使用100%的open gap penalty和0%的unit gap penalty,成对地计算该排列。使用0%的gap penalty,去除未知的碱基后,通过同源性计算形成相似性基质(similarity matrix)。使用邻接法(neighbor-joining method)构建结果树。
副球菌菌株R-1512的16SrDNA基因核苷酸序列被定义为SEQ ID NO12。R-1512及其亲缘关系最近的菌株之间的距离基质(distance matrix),作为16SrDNA序列相似性的百分数,如表5所示。菌株R-1512及其突变株衍生物R1534和R114的序列相同。R1506的序列与后面的菌株的序列仅一个核苷酸不同。这说明菌株R-1512和R-1506在系统发育学上高度相关,似乎属于相同的种(由DNA:DNA杂交证实,见下文)。R-1512和R-1506序列与那些可得的EMBL文库中序列所进行的对比,将R-1512和R-1506定位在副球菌属种。但是,观察到的、能够确凿地从所有目前的分类学角度说明副球菌种的序列相似性是小于97%,这个数值通常作为物种水平可能相关的极限值[Stackebrandt和Goebel,Int.J.Syst.Bacteriol.44846-849(1994)]。这说明R-1512菌株(及其突变衍生物)和R-1506属于一个或两个新的副球菌种。观察到四个未命名的副球菌菌株和菌株R-1512,R1534,R114和R-1506之间存在大于97%的序列相似性(在物种水平上可能的相关性显著),说明一个或多个未命名(MBIC)的菌株可能与菌株R-1512和R-1506在种水平上相关。根据簇分析(描述副球菌种菌株R-1512,R1534,R114,R-1506,MBIC3966以及副球菌属其它成员之间系统发育相关程度的系统发育树),菌株R-1512,R1534,R114和R-1506和四个未命名的副球菌菌株(MBIC3024,MBIC3966,MBIC4017和MBIC4020)被选择用于分析物种相关度的DNA:DNA杂交试验以分析物种的相关性。
表5.距离基质(distance matrix),作为副球菌种菌株R-1512及其亲缘关系最近的菌株之间16SrDNA序列相似性的百分数
a菌株类型后接aTDNA:DNA杂交及G+C含量测定用LMG培养基185培养表5所述细菌。根据Wilson的方法[In Ausabel等,(eds),Current Protocols in MolecularBiology,Greene Publishing and Wiley Interscience,New York,2.4.1-2.4.5(1987)],制备基因组DNA。根据Mesbach等人[Int.J.Syst.Bacteriol.39159-167(1989)],由Logan等人[Int.J.Syst.Bacteriol.501741-1753(2000)]进行的修改,利用HPLC进行DNA的G+C含量测定。报道的数值是对相同DNA样品进行测量得到的平均值。使用De Ley等人描述[Eur.J.Biochme.12133-142(1970)]的起始复性率方法[initial renaturation ratemethod],进行DNA:DNA杂交。杂交温度为81.5℃。Vauterin等人[Int.J.Syst.Bacteriol.45472-489(1995)]曾报道了该方法+/-5.8%的平均偏差。该细菌DNA的G+C含量以及DNA杂交试验结果归纳在表6中。
表6.副球菌种各菌株的DNA G+C含量(mol%),以及各菌株间DNA同源性百分比
a未测定菌株R-1512,R1534,R114,R-1506和MBIC3966之间DNA同源性大于70%(通常接受用于物种描述的极限值[Wayne等,Int.J.Syst.Bacteriol.37463-464(1987)],因而属于副球菌属中相同的种。这5个菌株的G+C含量在66.9%到67.7%之间,差别小于1%,表征着精确定义的物种。另一方面,菌株MBIC3024,MBIC4017及MBIC4020和R-1512,R1534,R114,R-1506及MBIC3966之间较低的DNA同源性说明,MBIC3024,MBIC4017及MBIC4020属于副球菌属内不同的基因种。
使用AFLPTM进行的DNA指纹AFLPTM是基于PCR技术,通过选择性扩增和选择性限制性片段显色[Vos等,Nucleic Acids Research234407-4414(1995);Janssen等,同上],对整个染色体DNA进行指纹印迹。该试验中,比较副球菌种菌株R-1512,R1534,R114,R-1506,MBIC3966和Paracoccus marcusii DSM 11574T,评价其种内相关度。在LMG培养基185中培养这些细菌。根据Wilson的方法(同上)制备这些细菌的中各个基因组DNA。用两种限制性酶——4-碱基酶切(cutter)酶和6-碱基酶切酶,消化纯化的DNA。通过这种方法,可以获得带有两种不同末端的、大小适于进行PCR的有限数目片段。含有一个相容性末端的接头(15-20bp的小双链DNA分子)与适当的限制性片段“粘性”末端相连。两个接头都是限制性半位点特异性,具有不同的序列。这些接头作为PCR引物的结合位点。本文中,所用的限制性酶是ApaI(6切的酶,识别序列GGGCC/C)和TaqI(4切的酶,识别序列T/GCA)。与用限制性酶酶切后形成的粘性末端相连的接头序列如表7所示(SEQ ID NO13-22)。使用PCR进行限制性片段的选择性扩增。该PCR引物特异性地与限制性片段的接头末端退火。由于引物的3’末端含有所谓的“选择性碱基”,这些碱基能够在限制性位点之外延伸到片段中,只有与限制性位点临近的、具备适当互补序列的限制性片段才能被扩增。所用的6个PCR引物序列组合也归纳在表7中。
表7.用于AFLPTM分析的接头和PCR引物
扩增后,使用DNA测序仪(ABI377),在高分辨率的聚丙烯酰胺凝胶上,根据各PCR产物的长度将其分离。由于相应引物5’末端标记了32P,含有限制性半位点(该位点由6-bp cutter形成)特异性接头的片段可通过放射自显影而进行目测。扫描电泳图案,用GelComparTM4.2软件(Applied Maths,B.V.B.A.,Kortrijk,Belgium)进行数字分析,使用Person曲线匹配系数(curvematching coefficient)和未加权的对群连接(unweighted pair group linking)分组[分组方法参见Sneath和Sokal,InNumerical Taxonomy.Freeman & Son,SanFrancisco(1973)]。
在所有的6个引物组合中(PCA-H,表7)副球菌属菌株R-1512,1534和R114的DNA指纹即使并非相同,也是高度近似。观察到微小区别的情况下,未估算重现性。曾预期这三种菌株的相似性或相同性会很高,因为R1534和R114来源于R-1512。运用所有的引物组合,菌株R-1512,1534和R114能够与菌株R-1506和MBIC3966清晰地区分开,后两种菌株均属于新的副球菌种。但是,指纹图谱并没有清楚地表明菌株R-1512,R1534和R114与R-1506或MBIC3966的亲缘关系更为密切。在所用的条件下,新物种聚簇(cluster)的五个菌株以大约58%(该值是六个AFLPTM试验(6个引物组合)中新物种分支点6个数值的平均数)相似性的平均水平进行分组,该组可以清楚地与Paracoccus marcusii DSM 11574T的图谱区分开,该典型菌株是系统发育学相关的产类胡萝卜素的副球菌种。Paracoccus marcusii DSM 11574T6个分支点与新物种的平均相似性值约为11%。
脂肪酸分析比较副球菌种菌株R-1512,R1534,R114,R-1506,MBIC3966与典型菌株P.marcusii DSM 11574T,P.carotinifaciens E-396T和P.solventivorans DSM 6637T的细胞膜脂肪酸组成。在28℃,用LMG培养基185培养细菌。使用商业系统MIDI(Microbial Identification System,Inc.,DE,USA),通过气相色谱测定脂肪酸组成。根据MIDI系统的建议进行脂肪酸的提取和分析。表8总结了所有受测菌株的结果。计算五个新副球菌种(R-1512,R1534,R114,R-1506,MBIC3966)菌株的平均图谱。所有8个有机体显示出可比较的细胞膜脂肪酸组成,181w7c是主要化合物。在新的副球菌种和三种典型菌株之间仅观察到微小的区别。
应用碳源进行生长为测试碳源的有氧应用,使用含有95种底物的BIOLOG-SF-N Microplate微量滴定板(Biolog Inc.,Hayward,CA,USA),只有E6孔的底物是D,L-乳酸甲酯,而不是通常所用的D,L-乳酸钠盐。在28℃下,表9所鉴定的每种菌株细胞均在LMG培养基12(Marine Agar,Difco 0979)中培养24小时。用无菌水制备密度等于0.5McFarland单位的细胞悬浮液。从该悬浮液中转移18滴液体到21ml的AUX培养基(API 20NE,bioMérieux,France)中,并轻轻混合。将0.1毫升悬浮液转移到BIOLOG MicroPlate的各孔中,30℃孵育该板。在48小时和6天后目测检查各孔的生长情况。同样,在第6天,使用BIOLOG板式读数器,通过阅读微量滴定板证实目测的分数。
BIOLOG分析的结果如表9。与没有底物的对照相比,不断增加的混浊度证实了生长(阳性反应)。在良好生长(+),微弱生长(±)和无生长(-)之间进行区别。括号内的结果是6天之后获得的,如果该值与48小时后获得的值不相符。问号表明第6天的结果不明确。在95种受测的碳源中,包括新的副球菌种(R-1512,R1534,R114,R-1506,MBIC3966)在内的所有五种菌株可以利用其中的12种用于生长,47种不能被利用。这5个菌株对剩下的36种底物表现出不同的生长反应。通过对7种碳源(福寿糖醇,i-赤藓糖醇,龙胆二糖,β-甲基葡糖苷,D-山梨糖醇,木糖醇和奎尼酸)的利用能力,新的副球菌种可以与两种其它的产类胡萝卜素细菌(P.marcusii DSM 11574T和P.carotinifaciens E-396T)相区分。新的副球菌种5种菌株都可利用的两种碳源(L-天冬酰胺和L-天冬氨酸)不能被P.marcusii DSM 11574T用于生长。
表8.副球菌种菌株R-1512,R1534,R114,R-1506,MBIC3966与其它副球菌种的三种典型菌株,即P.marcusii DSM 11574T,P.carotinifaciens E-396T和P.solventivorans DSM 6637T的细胞膜脂肪酸组成
aND,未检测。
生化试验使用API 20NE条带(strip)(bioMérieux,France)测试选出的生化特点。在28℃下,表10所鉴定的每种细菌菌株细胞培养于LMG培养基12中24小时。根据制造商的说明书制备细胞悬浮液及接种条带。条带(strip)在28℃下接种,在24和48小时后测定结果。结果总结在表10中。受测试的9个特点中,只有一个(尿素酶活性)特点在新副球菌中的5个菌株中有所区别。这9个特点在新副球菌种和P.marcusii DSM 11574T,P.carotinifaciensE-396T之间没有差异。
表9.副球菌种菌株利用碳源用于生长的情况
GalNAcN-乙酰-D-半乳糖胺;GluNAcN-乙酰基-D-葡糖胺;β-methylgucβ-甲基葡糖苷;MMSucc单-丁二酸甲酯;GalAlactoneD-半乳糖酸内脂;GalacturonicAD-半乳糖醛酸;GlucosaminicAD-葡糖甲酸(Glucosaminic acid);GlucuroinicAD-葡糖醛酸;AHBAα-羟基丁酸;BHBAα-羟基丁酸;GHBAγ-羟基丁酸;PHPAAp-羟基苯乙酸;AKBAα-丁酮酸;AKGAα-酮戊二酸;AKVAα-酮基戊酸;LAMED,L-乳酸甲酯;SaccAD-糖酸(saccharic acid);BromosuccA溴代丁二酸;GAA甘氨酰-L-天冬氨酸;GGA甘氨酰-L-谷氨酸;HydPro羟基-L-脯氨酸;PyroGluAL-焦谷氨酸;GABAγ-氨基丁酸;PEA苯乙胺;GlycPD,L-α-磷酸甘油;Gluc-1-P葡萄糖-1-磷酸;Gluc-6-P葡萄糖-6-磷酸表10.副球菌种菌株的生化特征12=R1512;34=R1534;14=R114;06=R1506;66=MBIC3966;74=DSM 11574T;96=E-396T;37=DSM 6637T
aβ-葡糖苷酶;b蛋白酶;S/+5Slow+5天生理试验对新副球菌种的5个菌株以及paracoccus marcusii DSM11574T,paracoccus carotinifaciens E-396T和paracoccus solventivorans DSM6637T进行几项理化和形态学试验。每项试验所用方法如下生长的温度范围 细胞在28℃,在LMG培养基12上培养24小时。用无菌水制备密度在1-2McFarland单位的细胞悬浮液。从该悬浮液中转移3滴液体到LMG培养基12的琼脂表面。一滴用划线法稀释,其余两滴不破坏。将该板在10℃,25℃,30℃,33℃,37℃和40℃下,孵育在有氧条件下,并在24、48小时和5天后检查生长情况。通过与30℃(即对照)下的生长情况相比较,可以目测其生长(各滴汇合,稀释的接种液在划的线上形成集落)。按下述打分(与对照板相比)生长优秀(++),生长较好(相当于对照)(+),生长较差(±),生长极差(±),无生长(-)。括号内结果是在划线处观察到的,其与保持未破坏液滴处的汇合生长情况不相同。
耐盐性 细胞在28℃,在LMG培养基12上培养24小时。用无菌水制备密度在1-2McFarland单位的细胞悬浮液。从该悬浮液中转移3滴液体到LMG培养基12的琼脂表面,所述培养基补充有NaCl,其终浓度为3%,6%和8%。一滴用划线法稀释,其余两滴未破坏。将该板在28℃下,温育在有氧条件下,并在24、48小时和5天后检查生长情况。通过与未加NaCl(对照)的生长情况相比较,可以目测其生长(各滴汇合,稀释的接种液在划的线上形成集落)。按下述打分(与对照板相比)生长优秀(++),生长较好(相当于对照)(+),生长较差(±),生长极差(±),无生长(-)。括号内结果是在划线处观察到的,其与保持未破坏液滴处的汇合生长情况不相同。
生长的pH范围 细胞在28℃,在LMG培养基12上培养24小时。用无菌水制备密度在1-2McFarland单位的细胞悬浮液。从该悬浮液中转移3滴液体到含10mlLMG培养基12液体培养基管中,所述培养基的pH值被调整,使灭菌后最终pH值为pH6.1,pH6.3,pH7.0,pH7.7,pH8.1和pH9.1。在28℃下有氧(振荡)培养该液体培养基。在24、48小时、第3天和第6天检查生长情况。通过与pH7.0(对照)的生长情况相比较,按增加的混浊度测定其生长情况(使用BIOLOG浊度计,按透射%进行测定)。按下述打分(与对照板相比)生长优秀(++),生长较好(相当于对照)(+),生长较差(±),生长极差(±),无生长(-)。
淀粉水解作用 细胞在28℃,在LMG培养基12平板上培养24小时。从平板上取一环细胞转移到补充有0.2%可溶性淀粉的LMG培养基12的琼脂表面,并划线。然后在28℃下有氧培养该液体培养基。当这些菌株达到生长良好状态时(48小时后),用鲁戈尔碘液(lugol solution)(0.5%I2和1%KI在蒸馏水中)覆盖该板。(淀粉未水解的区域琼脂的颜色为蓝色,与该区域对比)可以根据生长旁边的无色区域,测定水解程度,
反硝化作用 细胞在28℃,在LMG培养基12平板上培养24小时。从平板上取一环细胞插入含有半固体(0.1%琼脂)LMG培养基12的试管中,所述培养基补充有1%KNO3。在28℃下孵育该板5天。根据穿刺处旁边形成的气体测定脱氮作用(硝酸盐产生的N2)。
未加入电子接受体情况下,无氧条件下的生长细胞在28℃,在LMG培养基12平板上培养24小时。从平板上取一环菌转移到LMG培养基12的琼脂表面,并划线。在30℃、无氧条件(约10%CO2+约90%N2)下孵育该板。24小时后和5天后检查生长情况。通过与有氧条件(对照)相比较,测定其生长情况。按下述打分(与对照板相比)生长优秀(++),生长较好(相当于对照)(+),生长较差(±),生长极差(±),无生长(-)。
加入葡萄糖(发酵)情况下,无氧条件的生长 细胞在28℃,在LMG培养基12平板上培养24小时。从平板上取一环菌插入含有Hugh和Leifson[J.Bacteriol.6624-26(1953)]基础琼脂培养基的试管中。向培养基上部加入石蜡油,并在30℃孵育该板。48小时后和5天后检查生长情况。目测其生长情况。按下述打分生长好(+),生长差(±),无生长(-)。
以KNO3作为电子接受体,无氧条件下的生长情况 细胞在28℃,在LMG培养基12平板上培养24小时。从平板上取一环菌转移到补充有0.1%KNO3的LMG培养基12的琼脂表面,并划线。在30℃、无氧条件(约10%CO2+约90%N2)下孵育该板并在3天后检查生长情况。通过与有氧条件(对照)相比较,目测其生长情况。按下述打分(与对照板相比)生长优秀(++),生长较好(相当于对照)(+),生长较差(±),生长极差(±),无生长(-)。
过氧化氢酶和氧化酶反应 细胞在28℃,在LMG培养基12平板上培养24小时。过氧化氢酶活性的阳性结果表现为将菌落悬浮在一滴10%H2O2中,会有气泡产生。氧化酶活性的阳性结果表现为将菌落涂布在浸透了1%四甲基对苯二胺(tetramethylparaphenylene)的滤纸上,会形成紫红色。
菌落色素形成 细胞在28℃,在LMG培养基12上培养5天。肉眼观察菌落颜色。
细胞形态学和游动性 细胞在28℃,在LMG培养基12上培养24小时。用无菌水制备细胞悬浮液。使用配备有相衬光学(phase contrast optics)的Olympus光学显微镜(放大倍数1000x),观察细胞形态学和游动性。
表11总结了形态学和游动性试验的结果。新副球菌种的5个菌株对进行的所有理化或形态学试验中,反应基本相同。所有五个新副球菌种菌株反应相同、且能够使这些菌株与Paracoccus marcusii DSM 11574T和/或Paracoccus carotinifaciens E-396T相区分的试验是在40℃生长,在8%的NaCl中生长,pH9.1下生长和菌落色素沉着。
菌株R-1512,R1534,R114和R-1506的玉米黄质产量在ME培养基中培养R-1512,R1534,R114和R-1506菌株,所述培养基含有(每升蒸馏水)5g葡萄糖,10g酵母提取物,10g胰蛋白胨,30g NaCl和5g MgSO4·7H2O。高压灭菌前用5N NaOH将培养基的pH调整为7.2。所有的培养(25-ml体积在250ml带挡板、带塑料盖的Erlenmeyer烧瓶中)均在28℃下,200rpm振荡下进行。种子培养物是从冷冻的甘油化保藏物接种,并过夜培养。将等份种子培养物转移到试验烧瓶中,使其起始光学密度为660nm(OD660)下0.16。然后在28℃下,200rpm振荡培养。在整个培养过程检测生长情况,并在第6,10(或R114菌株时为15)和24小时,取出等份培养物,用实施例1所述方法分析类胡萝卜素。
这些条件下,菌株R-1512,R1534和R-1506的倍增时间分别为0.85小时,1.15小时和1.05小时。菌株R114再现性地显示出二相性的生长曲线;在初相菌株R114的倍增时间为1.4小时,而第二相中的倍增时间为3.2小时。
表12显示副球菌种菌株在ME培养基中的玉米黄质产量和特异性形成(将玉米黄质产量校正到OD660)。该数据是4种独立试验的平均值,其中每个试验的每个菌株均在两个烧瓶中进行试验。清楚显示出经典衍生的(classically-derived)突变株R1534和R114与亲代菌株R-1512进行比较的玉米黄质产量。菌株R-1506的玉米黄质产量与菌株R-1512大致相同。没有在任何培养物中检测到其他类胡萝卜素。
表11.副球菌种菌株的生理特性12=R1512;34=R1534;14=R114;06=R1506;66=MBIC3966;74=DSM 11574T;96=E-396T;37=DSM 6637T
Y-O黄色-橙色;O-P橙色-粉红色;PY浅黄;S-C短杆-球状;S短杆;C球状表12.副球菌种菌株R-1512,R1534,R114和R-1506的玉米黄质产量
实施例3在产玉米黄质的副球菌种菌株R114中通过甲羟戊酸途径进行的IPP生物合成。
为测定副球菌种菌株R114中类异戊二烯前体的生物合成(即甲羟戊酸或DXP途径)起源,采用“反生物合成(retrobiosynthesis)”方法[Eisenreich和Bacher,InSetlow(ed),Genetic Engineering,Principles and Methods,KluwerAcademic/Plenum Publishers,New York 22121-153(2000)]。这个预言性数据分析方法能够根据对单一下游天然产物的分析,准确地评估葡萄糖的分解代谢。本文中,该方法涉及在含有未标记葡萄糖和特定13C-标记葡萄糖的各种二元混合物的培养基中培养细菌,然后纯化生成的玉米黄质并通过NMR光谱学分析标记模式。下面详细介绍了所用方法以及试验结果。
培养用于13C标记试验的副球菌种菌株R114从Fluka(Milwaukee,WI,USA)购买未标记的D-葡萄糖一水化物。从Isotec(Miamisburg,OH,USA)购买[U-13C6]-D-葡萄糖,从Cambridge Isotope Laboratories(Andover,MA,USA)购买[1-13C1]-D-葡萄糖,[2-13C1]-D-葡萄糖和[6-13C1]-D-葡萄糖。从EMScience(Gibbstown,NJ,USA)购买酵母提取物和蛋白胨。所有的其它盐和溶剂为分析纯,购自正规化学试剂供应商。
所有的培养均来自冻冷的细胞悬浮液(细胞密度为12OD660单位,25%甘油,保藏在-70℃)。使用1ml解冻的细胞悬浮液,在含有100ml 362F/2培养基中,进行接种前培养(500ml带挡板的烧瓶),所述培养基含有以下组分30g/l D-葡萄糖,10g/l酵母提取物,10g/l蛋白胨,5g/l NaCl,2.5g/lMgSO4·7H2O,0.75g/l(NH4)2HPO4,0.625g/l K2HPO4,187.5mg/l CaCl2·2H2O,0.2g/l(NH4)2Fe(SO4)2·6H2O,15mg/l ZnSO4·7H2O,12.5mg/l FeCl3·6H2O,5mg/lMnSO4·H2O,0.5mg/l NiSO4·6H2O,15mg/l Na-EDTA和9.375μl/l HCl(37%储藏液)。该培养基的起始pH为7.2。
在28℃,200rpm振荡下进行24小时前培养,前培养之后,OD660约为22吸收单位。在含有362F/2培养基的Bioflo 3000生物反应器(NewBrunswick Scientific,Edison,NJ,USA)中进行主要培养,所述培养基含有以下组分30g/l总D-葡萄糖(见下面13C-标记未标记葡萄糖),20g/l酵母提取物,10g/l蛋白胨,10g/l NaCl,5g/l MgSO4·7H2O,1.5g/l(NH4)2HPO4,1.25g/l K2HPO4,0.4g/l(NH4)2Fe(SO4)2·6H2O,375mg/l CaCl2·2H2O,30mg/lZnSO4·7H2O,25mg/l FeCl3·6H2O,10mg/l MnSO4·H2O,1mg/l NiSO4·6H2O,30mg/l Na-EDTA和18.75μl/l HCl(37%储藏液)。四种试验中所用每种13C-标记葡萄糖数量(表达为培养基中总共30g/l葡萄糖的百分比)为条件1,4%[U-13C6]-D-葡萄糖;条件2,50%[1-13C1]-D-葡萄糖;条件3,25%[2-13C1]-D-葡萄糖+1%[U-13C6]-D-葡萄糖;条件4,25%[6-13C1]-D-葡萄糖+1%[U-13C6]-D-葡萄糖。同样包括未标记葡萄糖的对照。对于条件1和2(以及未标记的对照),培养体积为2l,而条件2和4为1l。用前培养物接种生物反应器(20ml/l初始体积),培养22-24小时,该时间点培养基内已没有葡萄糖剩余。培养条件为28℃,pH7.2(对照为25%H3PO4和28%NH4OH),溶解氧控制在(在格状物内搅拌(in a cascade with agitation))最小40%,搅拌速度和吹气速度分别为300rpm(最小)和1vvm。
纯化玉米黄质培养末期,将培养物冷却到15℃。向每升培养物中加入500ml无水乙醇(absolute ethanol),并以100rpm搅拌20分钟。将处理过的培养物在5000xg离心20分钟,弃上清。然后用5倍体积的THF搅拌提取湿沉淀20分钟。提取的混合物经离心后,保留上清,用1倍体积的THF在相同条件下再提取所获沉淀一次,离心。混合(提取物)上清,并通过旋转蒸发,浓缩到50ml。向浓缩的THF溶液中加入5ml己烷。混合后形成了乳状液,可通过离心将其分离。收集液体相,用等体积的饱和NaCl溶液稀释,用二氯甲烷再次提取。收集二氯甲烷相,并与THF/己烷相混合。然后通过旋转蒸发器再次浓缩该有机提取物的混合物,以去除二氯甲烷。然后将该溶液应用于硅胶柱,并用n-己烷和乙醚混合物(1∶1)洗脱。去除最先洗脱的一小条浅黄色带。主要的玉米黄质产物是在柱子中移动缓慢的一条宽带中被洗脱出来的。产物大约需要2升溶剂彻底地洗脱主要带。将洗脱液收集在圆底烧瓶中,并在40℃旋转蒸发去除溶剂。将残余物在40℃溶解于少量二氯甲烷中,然后使溶液慢慢冷却。向混合物中一滴一滴地加入己烷,直到出现混浊。在4℃下,48小时内彻底完成结晶。将结晶收集在滤纸上,用冷甲醇洗涤,真空干燥。
NMR研究NMR关谱学分析玉米黄质。为进行参考,以下式说明玉米黄质的化学结构 用Bruker DRX 500分光计,1H-NMR和13C-NMR光谱分别记录在500.13MHz和125.6MHz。一元试验和二元NADEQUATE试验的操作(acquisition)及处理参数均根据标准Bruker软件(XWINNMR)。溶剂氚化(deuterate)为氯仿。化学位移参照溶剂信号。
在相同条件下,记录同位素标记的玉米黄质样品的13C-NMR光谱,以及天然13C丰度(abundance)的玉米黄质样品的13C-NMR光谱。测定每个13C-NMR信号的积分(integrals),标记化合物中各个碳原子的信号积分与天然丰度材料的相参照,这样提供了标记分子物种中每个位点的相对13C丰度。然后在1.71ppm,H-18的1H NMR信号中,从13C偶联随体(couplingsatellite),将该相对丰度转化为绝对丰度。在多元标记的玉米黄质样品的13CNMR光谱中,每个随体被分别积分。然后将每个随体对的积分与指定碳原子的总信号积分相参照。玉米黄质含有总共八个类异戊二烯部分(2DMAPP单元和6IPP单元);仅仅观察到20个化学位移简并性的13C NMR信号。
用[U-13C6]葡萄糖和未标记葡萄糖混合物(1∶7.5;w/w)的试验中,玉米黄质的所有碳原子都被标记,并显示出13C13C偶联的随体(表13)。4个碳原子的信号具有高强度的13C13C偶联随体(在指定原子的总NMR信号强度中为61.2±0.6%,表13)。甲基原子C-17/C-17’的信号只显示出微弱的13C偶联随体,其相对强度为6%。中心的信号代表来自未标记葡萄糖的材料。这些信号未显示出远距离偶联的证据。很容易地从13C13C偶联常数(表13)和二元INADEQUATE试验发现碳的连通性(connectivity)。
三个碳原子获得了[6-13C1]葡萄糖的标记。其他两个碳原子被[2-13C1]葡萄糖标记。没有显著数量的标记来自[1-13C1]葡萄糖。
与未标记玉米黄质的光谱相比较,以及通过评价1H NMR光谱(表13)中1H13C偶联随体,来测定所有非等时碳原子的13C丰度。通过积分偶联随体,测定用[U-13C6]葡萄糖进行的试验中,连接转移的碳原子对所占部分。可根据重构建IPP前体所发现的标准偏差所示,精确地重构建IPP构建块的标记模式。DMAPP和IPP的重构建标记模式在试验极限内相同。
表13.提供了13C标记葡萄糖的副球菌种菌株R114所形成13C标记玉米黄质的NMR结果
以上测定的试验标记模式可以与各种预测相比,所述预测不仅考虑到用于类异戊二烯生物合成的甲羟戊酸途径与DXP途径,还考虑到不同的葡萄糖代谢途径。真细菌(Eubacteria)会典型地首先通过糖酵解代谢或Entner-Doudoroff途径利用葡萄糖。糖酵解会从葡萄糖产生两种丙糖磷酸分子。葡萄糖的C-1和C-7都转向了糖酵解过程中所形成的丙糖磷酸的3-位。另一方面,在Entner-Doudoroff途径中,葡萄糖转化为甘油醛3-磷酸和丙酮酸的混合物。葡萄糖的C-1无一例外地均转向丙酮酸的C-1,而葡萄糖的C-6则全部转向甘油醛3-磷酸的C-3。
糖酵解和Entner-Doudoroff途径的中间体和产物作为两种类异戊二烯生物合成途径的起始材料。关于甲羟戊酸途径,丙酮酸和丙糖磷酸可转化为乙酰-CoA的前体。通过糖酵解途径的葡萄糖代谢可将葡萄糖C-1和C-6的标记转到乙酰-CoA的甲基。通过Entner-Doudoroff途径的葡萄糖代谢可以在转化丙酮酸为乙酰-CoA的过程中,将葡萄糖的C-1损耗。
试验中观察到的副球菌种菌株R114所生成玉米黄质的富集及其13C13C偶联模式与Entner-Doudoroff途径和甲羟戊酸途径相结合时,玉米黄质生物合成所需要的标记模式完全一致。如果两个糖酵解和Entner-Doudoroff途径在所用试验条件下可同时进行,至少一些来自[1-13C1]葡萄糖的标记应该会进入玉米黄质。另外,甲羟戊酸途径可以最多将两个碳原子单元分配给类萜,而在DXP途径中,通过丙糖磷酸前体可将三个碳原子单元运送到类异戊二烯。尽管这样的三碳单元被DXP途径相关的重排所分离,三个标记碳原子的单元仍可通过远距离偶联识别。在培养的植物细胞(Cantharantusroseus)中,相应的13C-13C远距离偶联已在从[2,3,4,5-13C4]1-脱氧-D-木酮糖到类胡萝卜素叶黄素的生物合成中观察到[Arigoni等,Proc.Nat.Acad.Sci.9410600-10605(1997)]。副球菌种菌株R114形成的玉米黄质在本试验中未观察到这种长距离偶联。
应该注意,这里的结果证实了类异戊二烯通过甲羟戊酸途径在副球菌种菌株R114中形成,该结果还说明在所采用的生长条件下,几乎或完全没有糖酵解的葡萄糖代谢,并不排除在Entner-Doudoroff途径之外,通过戊糖磷酸途径进行一些葡萄糖代谢的可能性。可通过分析标记丙酮酸来源的氨基酸模式来定量测定以后两种途径进行的葡萄糖代谢(如对Paracoccusdenitrificans进行的过程[Dunstan等,Biomedical and Environ.MassSpectrometry 19369-381(1990)])。
实施例4副球菌种菌株R114中编码IPP异构酶和甲羟戊酸途径中各酶的基因的克隆及测序培养条件在28℃,F-培养基(10g/l胰蛋白胨,10g/l酵母提取物,30g/lNaCl,10g/l D-葡萄糖,5g/l MgSO4·7H2O,pH7.0)或在以上实施例3所述的前培养培养基中,培养副球菌种菌株R114。200rpm在旋转摇床上培养液体培养物。
分离基因组DNA将600ml副球菌种菌株R114的培养物在4℃,10000xg离心10分钟,用200ml溶解缓冲液(0.1M NaCl,50mM EDTA,10mMTris-HCl,pH7.5)洗涤沉淀一次,再用100ml溶解缓冲液洗涤一次。将最终获得的沉淀重悬浮于20ml含有50mg溶菌酶和1mg RNase A(不含DNase)的溶解缓冲液中。在37℃孵育15分钟后,加入1.5ml 20%N-月桂酰-肌氨酸钠和2.25mg蛋白激酶K。在50℃孵育30-60分钟后,用1倍体积的缓冲液-饱和苯酚,pH7.5-7.8(Life Technologies,Rockville,MD,USA),轻轻地但彻底地混合,提取溶解液。乳状液30000xg离心20分钟,用苯酚提取液体相。如前述分离该相并用1倍体积的苯酚∶氯仿(1∶1)提取两次。该步骤中,在翻转旋转器内3200xg离心20分钟,就可获得良好的相分离。用1倍体积的氯仿进行最后一次提取后,加入0.1倍体积3M乙酸钠(pH5.2),并用2倍体积冰冷的乙醇覆盖该溶液。用玻璃棒卷绕沉淀的DNA,浸入70%的乙醇5分钟,氯仿洗涤,然后干燥5-10分钟。将DNA重悬在5ml TE(10mMTris-HCl,pH7.5,1mM EDTA)中过夜。由于痕量的玉米黄质,该溶液为黄色,重复进行如上的有机提取和卷绕,直到获得无色的制剂。
λ-DNA的分离根据制造商的说明书,使用Qiagen_Lambda Kit(Qiagen,Hilden,Germany)。
聚合酶链式反应(PCR)从Life Technologies(Rockville,MD,USA)购买寡核苷酸。根据制造商的说明书,使用GC-rich PCR system(Roche MolecularBiochemicals,Mannheim,Germany),在GeneAmp_PCR system 9700(PEApplied Biosystems,Foster City,CA,USA)上进行PCR。通常,所用MgCl2浓度为1.5mM,加入溶解溶液的终浓度为1M。
DNA标记和检测分别使用PCR DIG Probe Synthesis Kit和DIGLuminescent Detection Kit(均购自Roche Molecular Biochemicals,Mannheim,Germany)进行DNA标记和检测。
DNA测序根据制造商的说明书,使用BigDye_DNA测序试剂盒(PEApplied Biosystems,Foster City,CA,USA)进行测序反应。测序反应在DyeExTM旋转柱(Qiagen,Hilden,Germany)上纯化,用ABI PrismTM 310Genetic Analyzer(PE Applied Biosystems,Foster City,CA,USA)进行片段分离和检测。
λ-文库用Sau3AI部分酶切的副球菌种菌株R114 DNA在λFIX_II中的定制文库购自Stratagene(La Jolla,CA,USA)。
副球菌种菌株R114甲羟戊酸途径基因簇的克隆,测序和表征甲羟戊酸途径各酶之一,甲羟戊酸二磷酸脱羧酶,含有高度保守的几个氨基酸长度的区域。从所有可获得的真细菌甲羟戊酸二磷酸脱羧酶的排列中选择三个这样的区域,使用在副球菌种菌株R1534类胡萝卜素基因簇中发现的偏爱密码子(表14)设计寡核苷酸。
为两个同源区域所设计的寡核苷酸如表15所示。为减低简并性程度,每个多肽均设计了几组寡核苷酸。例如,各个寡核苷酸mvd-103a-d仅在3’末端的第三个核苷酸有所区别,每各寡核苷酸均具有一种可能的甘氨酸密码子(尽管GGA很罕见,但还是将其容纳,因为其与3’末端紧密相连)。通过设计寡核苷酸为两个残基来说明可替换的氨基酸,如,在多肽1的第二个位置(表15),寡核苷酸mvd-101a和mvd-101b分别是亮氨酸或异亮氨酸特异的。使用副球菌种菌株R114DNA作为模板,用寡核苷酸mvd-101和mvd-104或mvd-106进行PCR,得到预期大小的产物。在载体pCR_2.1-TOPO(Invitrogen,Carlsbad,CA,USA)中克隆该PCR产物并测序。使用该克隆片段作为副球菌种菌株R114 DNA Southern分析的探针,发现其与一段约950bp的BamHI-SalI片段杂交。用BamHI和SalI酶切副球菌种菌株R114 DNA,琼脂糖凝胶电泳分离其片段。分离到约950bp的片段,并克隆到载体pUC19中。然后使用mvd-PCR片段作为探针,筛选该部分文库,测序插入的阳性克隆。并行地,使用mvd-PCR片段作为探针,筛选由副球菌种菌株R114 DNA制备的λ文库。从两个阳性λ克隆分离DNA,并用BamHI和SalI或EcoRI和SalI酶切。分离出大量的限制性片段,将其克隆到载体pUC19中。几个片段含有与编码甲羟戊酸途径各蛋白基因同源的序列。使用来自克隆的限制性片段各序列的引物,使用λ克隆的DNA作为模板,通过PCR,制备出连接这些独立序列的克隆。由所有片段(SEQ ID NO42,44,46,48,50和52)和被编码蛋白各序列组装而成的序列如序列表(SEQID NO43,45,47,49,51和53)所示。由于PatentIn Program的限制,重叠基因的操纵子不能作为单独的序列显示。这样,在甲羟戊酸操纵子中的各基因,每个基因操纵子的整个核苷酸序列均重复。相应地,SEQ ID NO42,44,46,48,50和52是相同的。为实现本发明,我们使用SEQ ID NO42指代甲羟戊酸操纵子的核苷酸序列。
饱和的或者不饱和的烃基,如烷基或者烯基,包括与杂原子连接的情况下,如在烷氧基中,只要可能,各为直链的或支链的。
除非特别指明,任选被取代的基团是被一取代或多取代的,其中在多取代的情况下,取代基可以相同或不同。
除了在制备实施例中提到的化合物外,可特别提及下述式(I-1-A-a)化合物。
X=4-C1-C6H4,Z=CH3。
表1
aSEQ ID NO1使用副球菌种菌株R1534偏爱密码子,见表12在一些酶中存在的可替换氨基酸S=C或G;R=A或G;Y=C或T;B=C或G或T;V=A或C或G通过基因产物与综合数据库中蛋白的同源性来鉴定副球菌种菌株R114的甲羟戊酸操纵子基因。用链霉菌种菌株CL190(SEQ ID NO54),S.griseolosporeus(SEQ ID NO55)和链霉菌种菌株KO-3899(SEQ ID NO56)的细菌I类HMG-CoA还原酶进行副球菌种菌株R114 HMG-CoA还原酶的氨基酸排列(SEQ ID NO43)。EMBL/GenBank/DDBJ数据库中,链霉菌种菌株CL190的登录号为q9z9n4,S.griseolosporeus的为q9znh1,链霉菌种菌株KO-3899的为q9znh0。存在两类HMG-CoA还原酶[Bochar等,Mol.Genet.Metab.66122-127(1999);Boucher等,Mol.Microbiol.37703-716(2000)]。真细菌的HMG-CoA还原酶通常属于第II类,而在真核生物和古细菌(archaea)中发现了I类酶。链霉菌和副球菌的HMG-CoA还原酶以及来自霍乱弧菌(Vibrio cholerae)的酶是目前所知仅有的真细菌I类HMG-CoA还原酶。
用EMBL数据库中找到的高度同源物,即草生欧文氏菌(Erwiniaherbicola)(Q01335)(SEQ ID NO57),布氏疏螺旋体(Borreliaburgdorferi)(O51627)(SEQ ID NO58),集胞蓝细菌种(Synechocystis)PCC6803(P74287)(SEQ ID NO59),链霉菌种CL190(Q9KWG2)(SEQ ID NO60),Streptomyces griseolosporeus(Q9KWF6)(SEQ ID NO61),硫磺矿硫化叶菌(Sulfolobus solfataricus)(P95997)(SEQ ID NO62),普氏立克次氏体(Rickettsia prowazekii)(Q9ZD90)(SEQ ID NO63),耐放射异常球菌(Deinococcus radiodurans)(Q9RVE2)(SEQ ID NO64),Aeropyrum pernix(Q9YB30)(SEQ ID NO65),盐杆菌属菌种(Halobacterium)NRC-1(O54623)(SEQ ID NO66),闪烁古生球菌(Archaeoglobus fulgidus)(O27997)(SEQ ID NO67),Pyrococcusabyssi(Q9UZS9)(SEQ ID NO68),Pyrococcus horikoshii(O58893)(SEQ ID NO69),嗜热碱甲烷杆菌(Methanobacterium thermoautotrophicum)(O26154)(SEQID NO70),詹氏甲烷球菌(Methanococcus jannaschii)(Q58272)(SEQ ID NO71),嗜酸热原体(Thermoplasma acidophilum)(CAC11250)(SEQ ID NO72),和Leishmania major(Q9NDJ5)(SEQ ID NO73),进行副球菌种菌株R114异戊烯二磷酸异构酶(IPP异构酶)(idi)的氨基酸排列(SEQ ID NO45)。EMBL/GenBank/DDBJ数据库会根据括号内的有机体名称,指定登录号。最先的9个序列来自真细菌,接下来的8个是来自古细菌(archaea)。有趣的是,一个真细菌物种,原生动物寄生虫Leishmania major(SEQ ID NO73)也具有一个高度同源的蛋白。这出乎意料,因为其它的真核生物具有不同的idi,命名为1型(Kaneda等,同上)。来自枯草杆菌的保守假定蛋白YpgA,也具有较高的同源性,但比2型idi要小很多。
用EMBL数据库中找到的高度同源物,即肺炎链球菌(Streptococcuspneumoniae)(AAG0245 3)(SEQ ID NO74),Streptococcus pyrogenes(AAG02448)(SEQ ID NO75),粪肠球菌(Entereococcus faecalis)(AAG02438)(SEQ ID NO76),屎肠球菌(Entereococcus faecium)(AAG02443)(SEQ ID NO77),溶血葡萄球菌(Staphylococcus haemolyticus)(AAG02427)(SEQ ID NO78),表皮葡萄球菌(Staphylococcus epidermis)(AAG02433)(SEQ ID NO79),金黄色葡萄球菌(Staphylococcus aureus)(AAG02422)(SEQ ID NO80),肉葡萄球菌(Staphylococcus carnosus)(Q9ZB67)(SEQ ID NO81),链霉菌种CL190(Q9KWG1)(SEQ ID NO82),Streptomyces griseolosporeus(Q9KWF5)(SEQ ID NO83),和布氏疏螺旋体(051626)(SEQ ID NO84),进行副球菌种菌株R114细菌HMG-CoA合成酶的氨基酸排列(SEQ ID NO47)。EMBL/GenBank/DDBJ数据库会根据括号内有机体的名称,指定登录号。
使用来自其它细菌的直向同源(orthologous)蛋白,即,肺炎链球菌(AAG02456)(SEQ ID NO85),Streptococcus pyrogenes(AAG02451)(SEQ IDNO86),粪肠球菌(AAG02441)(SEQ ID NO87),屎肠球菌(AAG02446)(SEQ ID NO88),溶血葡萄球菌(AAG02431)(SEQ ID NO89),表皮葡萄球菌(AAG02436)(SEQ ID NO90),金黄色葡萄球菌(AAG02425)(SEQ ID NO91),链霉菌种CL190(Q9KWG4)(SEQ ID NO92),Streptomycesgriseolosporeus(Q9KWF8)(SEQ ID NO93),和布氏疏螺旋体(051629)(SEQID NO94),进行副球菌种菌株R114细菌甲羟戊酸二磷酸脱羧酶的氨基酸排列(SEQ ID NO53)。EMBL/GenBank/DDBJ数据库会根据括号内有机体的名称,指定登录号。
黄色粘球菌(Myxococcus xanthus)的两种蛋白,Tac和Taf(数据库登录号分别为q9xb06和q9xb03)以及枯草杆菌蛋白PksG(一种假定的多聚乙酰生物合成蛋白(数据库登录号为p40830)),与副球菌种菌株R114 HMG-CoA合成酶具有较高的同源性。副球菌种菌株R114 HMG-CoA合成酶与黄色粘球菌Tac和Taf蛋白的同源性高于副球菌种菌株R114 HMG-CoA合成酶与真核生物的同源性。细菌HMG-CoA合成酶和细菌甲羟戊酸二磷酸脱羧酶与其真核生物的直向同源物具有基本的特异性。古细菌(archaeal)HMG-CoA合成酶组成了一个亲缘较远的酶组(Wilding等,同上),而在古细菌内未找到甲羟戊酸二磷酸脱羧酶的直向同源物[Smit and Mushegian,Genome Res.101468-1484(2000)]。
使用来自其它细菌的直向同源(orthologous)蛋白,即,肺炎链球菌(AAG02455)(SEQ ID NO95),Streptococcus pyrogenes(AAG02450)(SEQ IDNO96),粪肠球菌(AAG02440)(SEQ ID NO97),屎肠球菌(AAG02445)(SEQ ID NO98),溶血葡萄球菌(AAG02430)(SEQ ID NO99),表皮葡萄球菌(AAG02435)(SEQ ID NO100),金黄色葡萄球菌(AAG02424)(SEQ ID NO101),链霉菌种CL190(Q9KWG5)(SEQ ID NO102),Streptomyces griseolosporeus(Q9KWF9)(SEQ ID NO103),和布氏疏螺旋体(051631)(SEQ ID NO104)(Mvk),和,抗肺炎链球菌(AAG02457)(SEQID NO105),Streptococcus pyrogenes(AAG02452)(SEQ ID NO106),粪肠球菌(AAG02442)(SEQ ID NO107),屎肠球菌(AAG02447)(SEQ ID NO108),溶血葡萄球菌(AAG02432)(SEQ ID NO109),表皮葡萄球菌(AAG02437)(SEQ ID NO110),金黄色葡萄球菌(AAG02426)(SEQ ID NO111),链霉菌种CL190(Q9KWG3)(SEQ ID NO112),Streptomycesgriseolosporeus(Q9KWF7)(SEQ ID NO113),和布氏疏螺旋体(051630)(SEQID NO114)(pmk),进行副球菌种菌株R114的甲羟戊酸激酶(Mvk)(SEQ IDNO49)和磷酸甲羟戊酸激酶(Pmk)(SEQ ID NO51)排列。EMBL/GenBank/DDBJ数据库会根据括号内有机体的名称,指定登录号。
细菌激酶之间的同源性要远远低于甲羟戊酸途径其它酶细菌直向同源物之间的同源性。副球菌种菌株R114的甲羟戊酸激酶(SEQ ID NO49)在氨基末端区域内具有一个37个氨基酸长度的插入,这在其它甲羟戊酸激酶中是不存在的。细菌Mvk以及一些古细菌酶,如来自闪烁古生球菌(Archaeoglobus fulgidus),嗜热碱甲烷杆菌和Pyrococcus abyssi的酶,是与副球菌种菌株R114的Mvk同源性最高的酶。细菌磷酸甲羟戊酸激酶之间的同源性甚至低于细菌甲羟戊酸激酶之间的同源性。与副球菌种菌株R114Pmk(SEQ ID NO51)同源性最高的蛋白是来自古细菌的Mvk,如Aeropyrumperuix,Pyrococcus horikoshii,M.thermoautotrophicum,P.abyssi和A.fulgidus。由于在古细菌中未发现Pmk(Smit and Mushegian,同上),这说明在两种磷酸化过程中可能是同一种激酶在起作用。
实施例5副球菌种菌株R114甲羟戊酸途径各基因和idi基因在大肠杆菌的过量表达甲羟戊酸操纵子在大肠杆菌中的克隆和表达一个λ克隆,命名为克隆16,来自副球菌种菌株R114的λ文库(见实施例4),将其作为PCR扩增甲羟戊酸操纵子的模板。PCR所用引物为Mevop-2020和Mevop-9027(表16)。
表16.扩增副球菌种菌株R114甲羟戊酸操纵子所用引物
将所获的PCR产物克隆到TOPO-XL(Invitrogen,Carlsbad,CA,USA)中,得到质粒TOPO-XL-mev-op16。插入的部分带有甲羟戊酸操纵子,用HindIII和SacI酶切该插入部分,并克隆到HindIII-SacI处理过的载体pBBRlMCS2[Kovach等,Gene 166175-176(1995)]中,得到了质粒pBBR-K-mev-op16。使用质粒pBBR-K-mev-op16转化电穿孔-感受态大肠杆菌菌株TG1[Stratagene,La Jolla,CA;Sambrook等,InNolan,C.(ed),Molecular CloningA Laboratory Manual(Sencond Edition),p.A.12(1989)]。将两个代表性的阳性转化株(E.coli TG1/pBBR-K-mev-op16-1和E.coliTG1/pBBR-K-mev-op16-2)培养在含有50mg/l卡那霉素的Luria Broth(LB,GibcoBRL,Life Technologies)中,并使用实施例1所述方法,检测HMG-CoA还原酶活性(由副球菌种菌株R114的mvaA基因编码)。大肠杆菌并不具有编码HMG-CoA还原酶的基因,因此缺乏可检测的活性。E.coliTG1/pBBR-K-mev-op16两个代表性转化株的粗提物具有可简易测定的HMG-CoA还原酶活性,说明克隆的mvaA基因得到了异源表达。
表17.携带了克隆的副球菌种菌株R114甲羟戊酸基因簇的大肠杆菌TG1细胞粗提物中HMG-CoA还原酶活性
a小于0.03U/mg副球菌种菌株R114 idi基因和甲羟戊酸途径单个基因在大肠杆菌中的克隆与表达使用表18所示引物,PCR扩增副球菌种菌株R114甲羟戊酸操纵子各基因的编码区域。引物的设计使得ATG起始密码子构成了NdeI位点的后半(second)部分(切割识别位点CATATG),而在终止密码子后随即导入BamHI位点(GGATCC)。所有的PCR产物均克隆到pCR_2.1-TOPO载体中。所获载体的名字列在表19中。除了甲羟戊酸激酶基因,所有的基因都含有BamHI,NdeI或EcoRI的限制性位点,这些位点可以被消除,以简化后面的克隆步骤。可使用QuikChangeTM定点突变试剂盒(Strategene,La Jolla,CA,USA)和表20所示寡核苷酸,通过导入沉默突变消除这些位点。用BamHI和NdeI从TOPO质粒中切除突变的编码区域,并与BamHI-NdeI酶切的表达载体pDS-His和pDS相连。这些表达载体均来自pDSNdeHis,该载体在EP 821,063的实施例2中有所描述。通过缺失一段857个bp的NheI和XbaI片段(该片段带有沉默的氯霉素乙酰基转移酶基因),从pDSNdeHis构建质粒pDS-His。质粒pDS是通过用退火的引物S/D-1(5’AATTAAAGGAGGGTTTCATATGAATTCG)(SEQ ID NO117)和S/D-2(5’GATCCGAATTCATATGAAACCCTCCTTT)(SEQ ID NO118)替换一小段EcoRI-BamHI片段,从pDS-His构建而来。
表18.克隆甲羟戊酸操纵子基因的寡核苷酸
1第二个密码子TCA改为AGC(沉默突变-两个密码子均编码丝氨酸)。
2最后密码子GGC改为GGA(沉默突变-两个密码子均编码甘氨酸)。
表19.表达质粒的名称和构建中间体
n/a不适用;nd未作在36小时发酵期间产生63.8g L-苏氨酸。在此实验中,产率为1.77g/h。
表11营养培养基M1-160的组分
表12生长培养基M1-546的组分
实施例5借助于菌株大肠杆菌K-12菌株B-3996,通过5次随后的补料方法及在每种情况使用10%接种物制备L-苏氨酸表21.过量表达来自副球菌种菌株R114的克隆mvaA基因的大肠杆菌菌株体内HMG-CoA还原酶活性的诱导
aM15/pDS-mvd作为阴性对照表22.过量表达来自副球菌种菌株R114的克隆idi基因的大肠杆菌菌株体内IPP异构酶活性的诱导接92页表22
aM15/pDS-mvd作为阴性对照b<1U/mg通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE),分析用于酶试验的粗提物。对于菌株E.coli M15/pDS-mvaA和E.coli M15/pDS-His-mvaA,预期分子量(36.3kD)蛋白的高度表达与否是与粗提物中所测的HMG-CoA还原酶活性(表21)相关联的。可这样解释His-标记蛋白的缺乏mRNA或蛋白的不稳定性降低了转录或翻译水平的表达。E.coli M15/pDS-idi和E.coliM15/pDS-His-idi的粗提物分别显示出预期分子量37.3kD和39.0kD蛋白的高度表达。但是,只有E.coli M15/pDS-idi的IPP异构酶活性提高(表22),说明该酶的组氨酸标记形式在这些条件下不起作用。
通过对过量表达其它四种副球菌种菌株R114甲羟戊酸操纵子基因(hcs,pmk,mvk和mvd,参照表19)的大肠杆菌菌株粗提物的SDS-PAGE分析,IPTG诱导后,并未检测到该酶天然形式的高表达,尽管有些表达不能被排除。另一方面,均观察到所有四种His-标记蛋白的高表达。
实施例6通过过量表达crtE基因,改进副球菌种菌株R114的玉米黄质产量构建pBBR-K-Zea4,pBBR-K-Zea4-up和pBBR-K-Zea4-down以及这些质粒对副球菌种菌株R114玉米黄质产量的影响从质粒pZea-4质粒中切出一段8.3kb BamHI-EcoRI片段,该片段为副球菌种菌株R1534的类胡萝卜素(crt)基因组簇。将含有crt基因的该片段连接到BamHI和EcoRI酶切过的载体pBBR1MCS-2(GenBank登录号#U23751)中,得到pBBR-K-Zea4。通过连接,将质粒pBBR-K-Zea4导入副球菌种菌株R114中,以检测其改进的玉米黄质产量。在摇瓶培养基(使用362F/2培养基,见实施例11)中,测定对照菌株R114和R114/pBBR-K-Zea4的两个独立分离菌株玉米黄质产量。表23中的数据显示携带质粒pBBR-K-Zea4的两个重组菌株玉米黄质产量显著高于R114,且生产的比速率(specific rate)也更高(mg玉米黄质/OD660)。这说明在pBBR-K-Zea4的克隆插入片段中有一个或多个基因编码着限制副球菌种菌株R114玉米黄质产量的酶。
表23.菌株R114和R114/pBBR-K-Zea4的玉米黄质产量
a玉米黄质b比形成(specific formation)(mg ZXN/1/OD660)为定位阳性作用,制备两种衍生质粒,使其含有pBBR-K-Zea4中存在的克隆插入片段的亚克隆区域。该pBBR-K-Zea4插入含有ORF 5和基因atoB和crtE(Pasamontes等,同上),使该插入的“上游”区域被限制性酶XbaI和AvrII唯一位点侧接。用这两种酶酶切消化pBBR-K-Zea4并缺失“上游”区域,构建质粒pBBR-K-Zea4-down。类似地,使用限制性酶EcoRI和StuI,在pBBR-K-Zea4的克隆插入片段内缺失“下游”区域,构建质粒pBBR-K-Zea4-up。通过接合,将这两个新的质粒转移到副球菌种菌株R114中。比较(摇瓶培养,与前述相同的条件)菌株R114(宿主对照(host control)),R114/pBBR-K(空白载体对照),R114/pBBR-K-Zea4-down和R114/pBBR-K-Zea4-up的玉米黄质产量(表24)。该数据清楚地表明对玉米黄质产量的阳性作用是存在克隆片段多个拷贝的结果,所述片段含有ORF5,atoB和crtE,即是质粒pBBR-K-Zea4-up中存在的插入片段。从pBBR-K-Zea4-up构建一系列缺失质粒。将这些质粒逐一导入菌株R114并测定玉米黄质产量,结果表明过量表达crtE基因引发了菌株R114/pBBR-K-Zea4和pBBR-K-Zea4-up玉米黄质产量的提高。该结果与副球菌种菌株R114体内限制玉米黄质产量的GGPP合成酶(由crtE编码)活性相一致。使用实施例1所述方法,发现R114/pBBR-K-Zea4-up粗提物的GGPP合成酶活性比R114高2.6倍。为直接证实改结果,按下述两部分,构建能够且仅仅能够过量表达crtE基因的新质粒。
表24.带有pBBR-K-Zea4质粒缺失衍生物的菌株中玉米黄质产量
a玉米黄质b比形成(specific formation)(mg ZXN/1/OD660)
构建表达载体pBBR-K-PcrtE和pBBR-tK-PcrtE用BstXI和Bsu36I酶切载体pBBR1MCS-2,将较大的片段与退火的寡核苷酸MCS-2上游(5’TCAGAATTCGGTACCATATGAAGCTTGGATCCGGGG3’)(SEQ ID NO145)和MCS-2下游(5’GGATCCAAGCTTCATATGGTACCGAATTC3’)(SEQID NO146)相连,得到载体pBBR-K-Nde。副球菌种菌株R114中类胡萝卜素基因簇的crtE基因的270bp区上游片段含有假定的crtE启动子(PcrtE),该启动子含有核糖体结和位点和crtE起始密码子(Pasamontes等,同上),通过PCR,使用引物crtE-up(5’GGAATTCGCTGCTGAACGCGATGGCG3’)(SEQ ID NO147)和crtE-down(5’GGGGTACCATATGTGCCTTCGTTGCGTCAGTC)(SEQ ID NO148),从副球菌种菌株R114中扩增该上游片段。用EcoRI和NdeI酶切该PCR产物,并插入EcoRI-NdeI酶切过的pBBR-K-Nde骨架,形成质粒pBBR-K-PcrtE。在引物crtE-down中包含一个NdeI位点,该位点含有crtE的起始密码子ATG。因此,任何被导入的、含有存在于NdeI位点中的起始密码子的编码区域,都可以使用crtE的核糖体结合位点被表达。用BamHI酶切pBBR-K-PcrtE,并插入退火的寡核苷酸pha-t-up(5’GATCCGGCGTGTGCGCAATTTAATTGCGCACACGCCCCCTGCGTTTAAAC3’)(SEQ ID NO149)和pha-t-down(5’GATCGTTTAAACGCAGGGGGCGTGTGCGCAATTAAATTGCGCACACGCCG3’)(SEQ ID NO150)。测序验证插入的片段,将下述的质粒命名为pBBR-tK-PcrtE寡核苷酸插入的方向使得重组成的BamHI位点靠近PcrtE启动子。该插入序列带有在副球菌种菌株R114 phaA和phaB基因之间发现的(见实施例10)、假定的转录终止子,因此该序列应该能够适当地终止从PcrtE启动子开始的转录。
构建质粒pBBR-K-PcrtE-crtE-3为构建多元拷贝质粒用于提高crtE基因在副球菌种菌株R114宿主中的表达,使用引物crtE-Nde(5’AAGGCCTCATATGACGCCCAAGCAGCAATT3’J)(SEQ ID NO151)和crtE-Bam(5’CGGGATCCTAGGCGCTGCGGCGGATG3’)(SEQ ID NO152),从质粒p59-2(Pasamontes等,同上)中扩增crtE基因。将扩增的片段克隆到载体pCR_2.1-TOPO中,得到质粒TOPO-crtE。将TOPO-crtE的NdeI-BamHI片段亚克隆到NdeI-BamHI酶切过的质粒pBBR-K-PcrtE中,得到pBBR-K-PcrtE-crtE。最后,用pBBR-K-Zea4-up的较小BglII片段替换pBBR-K-PcrtE-crtE的较小BglII片段,构建pBBR-K-PcrtE-crtE-3。通过电穿孔,将质粒pBBR-K-PcrtE-crtE-3转移到副球菌种菌株R114中。使用实施例1所述方法,测定R114/pBBR-K-PcrtE-crtE-3粗提物中GGPP合成酶活性比菌株R114高2.9倍。活性提高程度类似于在R114/pBBR-K-Zea4-up观察到的结果。表25显示菌株R114/pBBR-K-PcrtE-crtE-3玉米黄质测量与R114/pBBR-K-Zea4-up基本相同。
表25.比较菌株R114/pBBR-K-PcrtE-crtE-3和R114/pBBR-K-Zea4-up的玉米黄质产量
a玉米黄质b比形成(specific formation)(mg ZXN/1/OD660)实施例7副球菌种菌株R114甲羟戊酸操纵子各独立基因在天然宿主副球菌种菌株R114中的表达副球菌种菌株R114甲羟戊酸操纵子各独立基因在副球菌种菌株R114宿主中的表达用BamHI和NdeI酶切TOPO-质粒中突变的甲羟戊酸操纵子基因编码区域(见实施例5),并与BamHI-NdeI酶切过的载体pBBR-tK-PcrtE-crtE(见实施例6)相连。通过电穿孔,将获得的质粒pBBR-tK-PcrtE-mvaA,pBBR-tK-PcrtE-idi,pBBR-tK-PcrtE-hcs,pBBR-tK-PcrtE-mvk,pBBR-tK-PcrtE-pmk和pBBR-tK-PcrtE-mvd导入副球菌种菌株R114中。在含有50mg/l卡那霉素的琼脂培养基上选择转化株,并PCR验证。
为说明由质粒提供的甲羟戊酸途径各基因可在天然宿主副球菌种菌株R114内表达,比较菌株R114/pBBR-K(对照)和R114/pBBK-tK-PcrtE-mvaA粗提物的HMG-CoA还原酶活性(所用方法如实施例1所述)。菌株R114/pBBR-K和R114/pBBK-tK-PcrtE-mvaA的HMG-CoA比活性分别为2.37U/mg和6.0U/mg。这样在多元质粒上存在(以及从PcrtE启动子表达)mvaA基因,会使HMG-CoA还原酶活性由带有空载体pBBR-K的R114基础(即,染色体编码的)活性提高2.5倍。
实施例8构建“Mini-操纵子”用于同时过量表达甲羟戊酸途径的克隆基因和副球菌种菌株R114的crtE基因质粒构建如实施例6所示,将质粒pBBR-K-PcrtE-crtE-3导入副球菌种菌株R114,引起玉米黄质产量提高,说明GGPP合成酶活性是菌株R114中玉米黄质生物合成的限速因素。实施例7进一步显示了编码甲羟戊酸途径各酶的基因可在天然宿主副球菌种菌株R114中过量表达,导致各编码酶活性提高。但是,携带着质粒(所述质粒含有甲羟戊酸操纵子各个独立基因)的副球菌种菌株R114重组菌株均未显示出提高的玉米黄质产量。可能是玉米黄质途径中下游的“瓶颈”(GGPP合成酶)屏蔽了甲羟戊酸途径操纵子各基因在副球菌种菌株R114中过量表达所带来的益处。制备出能够同时过量表达甲羟戊酸途径各个基因(或这些基因的组合)和crtE的质粒可以释放整个玉米黄质生物合成途径中的所有限速步骤,从而提高玉米黄质产量。下面的部分描述了“Mini-操纵子”的构建,用于使crtE和编码甲羟戊酸途径5种酶的各个基因共过量表达。
使用BamHI和NdeI酶,从各个TOPO-质粒(实施例5和6中所述)中切出crtE,mvaA,idi和mvk基因,并与BamHI-NdeI酶切过的载体pOCV-1(实施例12所述)连接。crtE基因编码区域的最后核苷酸并非腺嘌呤,而且,具有TAG而不是TGA终止密码子,终止密码子和BamHI位点之间的距离不合适。因此,crtE的末端并不符合操纵子构建载体的要求(参见实施例12),而且crtE必须是用pOCV-1-crtE构建的任何操纵子中的最后一个基因。为满足将腺嘌呤作为第二个密码子的第一个核苷酸、且作为最后密码子的最后一个核苷酸的条件,必须在甲羟戊酸操纵子的三个基因中引入突变。pmk的第二个密码子,GAT,编码Asp,被改变为编码Asn的AAT。mvd的最后密码子以T结尾,而pmk和hcs以C结尾。将这些核苷酸突变为A,引起沉默突变,pmk除外(pmk的最后氨基酸由Asp变为Glu)。设计寡核苷酸,通过PCR导入必要的改变。这些PCR反应所用的寡核苷酸序列和模板列在表26中。将所有的PCR产物均克隆到pCR_2.1-TOPO载体中,形成质粒TOPO-mvdOCV,TOPO-pmkOCV和TOPO-hcsOCV。用NdeI和BamHI酶切插入的部分,并与NdeI-BamHI尘粒过的pOCV-2(见实施例2)相连。组装各个“Mini-操纵子”的最后克隆步骤与前述类似,可由构建pBBR-K-PcrtE-mvaA-crtE-3的代表性方案说明。
表26.构建质粒TOPO-mvdOCV,TOPO-pmkOCV和TOPO-hcsOCV的PCR所用寡核苷酸和模板
实施例9副球菌种菌株R114中编码FPP合成酶的ispA基因的克隆和测序由于FPP合成酶存在在副球菌种菌株R114玉米黄质生物合成中的中间途径中,通过提高ispA基因的量从而提高该酶活性的做法对于改进玉米黄质的产量十分有效。因此,按下述克隆并测序副球菌种菌株R114的ispA基因。从公共数据库中获得6种细菌FPP合成酶的氨基酸序列。这些序列具有几个高度保守的区域。表27列出了这些区域中的两个以及PCR所用寡核苷酸。使用副球菌种菌株R114 DNA作为模板,与寡核苷酸GTT-1和GTT-2进行PCR,形成了预期大小的产物。将该PCR产物克隆到载体pCR_2.1-TOPO中并测序。使用克隆的片段作为副球菌种菌株R114 DNASouthern分析中的探针,发现其与一段大约1.9kb的BamHI-NcoI片段杂交。用BamHI和NcoI酶切副球菌种菌株R114 DNA,琼脂糖凝胶电泳分离各片段。在15.-2.1kb之间的区域被分离,并克隆到克隆载体的BamHI和NcoI位点。然后使用ispA-PCR片段作为探针,筛选该部分文库,分离得到两个阳性克隆。测序结果证实了两个克隆质粒均含有ispA基因。ispA基因(SEQID NO159)的上游是核酸外切酶VII的小亚基,XseB(SEQ ID NO158),下游是编码1-脱氧木酮糖-5-磷酸合成酶的dxs基因(SEQ ID NO160)。这与大肠杆菌中发现的基因排列相同。NcoI-BamHI片段序列如SEQ ID NO157所示,XseB,IspA和Dxs的氨基酸序列分别如SEQ ID NO158,SEQ ID NO159和SEQ ID NO160所示。ispA的起始密码子可以是GTG或ATC,其可在天然IspA的氨基末端,分别形成两个或一个甲硫氨酸残基。
使用实施例5-7所述的常规克隆策略,可以构建一个新的质粒,pBBR-tK-PcrtE-2,其能够在天然宿主副球菌种菌株R114中过量表达ispA基因。利用电穿孔,将该质粒导入菌株R114,并PCR验证转化株。在362F/2培养基(实施例11)上培养三种具有代表性的转化株和对照菌株(R114/pBBR-K),制备粗提物,并根据实施例1所述方法,试验测定ispA基因产物FPP合成酶的活性。菌株R114/pBBR-K中基础(染色体编码的)FPP合成酶比活性为62.6U/mg。三种转化株中FPP合成酶的比活性为108.3U/mg(增长73%),98.5U/mg(增长57%)和83.8U/mg(增长34%),说明在副球菌种菌株R114中,以活性形式过量表达ispA基因并且过量生成其产物FPP合成酶。
表27.用于两种保守的细菌IspA基因多肽的寡核苷酸
Y=C或T实施例10副球菌种菌株R114中编码乙酰-CoA乙酰基转移酶各基因的克隆和测序IPP生物合成中第一个进行的步骤是利用HMG-CoA合成酶,将乙酰-CoA和乙酰乙酰-CoA缩合为羟基甲基戊二酸单酰-CoA(HMG-CoA)。底物乙酰乙酰-CoA是通过乙酰-CoA乙烯基转移酶(又称为乙酰乙酰-CoA硫解酶或β-酮硫解酶),缩合两分子乙酰-CoA而形成。由于该反应通过甲羟戊酸途径,将中间代谢(乙酰-CoA处)和类异戊二烯的生物合成相连接,因此,通过基因扩增提高乙酰-CoA乙酰基转移酶的活性从而提高体内的碳流向类胡萝卜素和其它类异戊二烯的方法十分有效。在副球菌种菌株R114中,至少有两种基因——atoB和phaA——编码乙酰-CoA乙酰基转移酶。副球菌种菌株R1534(US 6,087,152)和R114(本文)中,atoB基因的末尾位于crtE起始部位上游的165个核苷酸处。副球菌种菌株R1534中,被编码的乙酰-CoA乙酰基转移酶的atoB基因核苷酸序列和相应的氨基酸序列分别如SEQ IDNO175和SEQ ID NO176所示。
使用实施例5所述的常规克隆策略,atoB基因被克隆到质粒pDS和pDS-His中。将新的质粒——pDS-atoB和pDS-His-atoB——导入大肠杆菌M15。在具有IPTG诱导(如实施例5所述)和不具有IPTG诱导的条件下,培养获得的菌株M15/pDS-atoB和M15/pDS-His-atoB,制备粗提物用于乙酰-CoA乙酰基转移酶试验(方法如实施例1所述)和SDS-PAGE分析。在M15/pDS-atoB和M15/pDS-His-atoB(带诱导)的粗提物中,乙酰-CoA乙酰基转移酶的比活性分别为0.2U/mg和13.52U/mg。在不含质粒的大肠杆菌中测得的基础活性为0.006U/mg。IPTG诱导后,atoB基因产物——乙酰-CoA乙酰基转移酶在大肠杆菌M15中过量生成。天然(M15/pDS-atoB)和His-标记的(M15/pDS-His-atoB)形式中均过量生成。M15/pDS-His-atoB过量生成的程度要高得多,该结果与两个(诱导的)菌株粗提物中所测到的乙酰-CoA乙酰基转移酶活性一致。
乙酰乙酰-CoA还是聚羟基烷羧酸酯(PHA)生物合成所进行的第一个步骤中的底物。在许多细菌中,与PHA生物合成相关的基因都被分入各个操纵子的组中[Madison和Huisman,Microbol.Mol.Biol.Rev.,6321-53(1999)]。在Paracoccus denitrificans中,phaA和phaB基因,分别编码乙酰-CoA乙酰基转移酶和乙酰乙酰-CoA还原酶,这两个基因成簇于同一个操纵子中[Yabutani等,FEMS Microbiol.Lett.13385-90(1995)],而编码该途径最后一个酶——聚(3-烷羧酸酯)(3-hydroxyalkalkanate)合成酶——的phaC则不属于该操纵子[Ueda等,J.Bacteriol.178774-779(1995)]。使用根据P.denitrificansphaA和phaC基因序列合成的引物,获得含有副球菌种菌株R1534 phaA部分和副球菌种菌株R114 phaC的PCR片段。然后将该PCR片段作为探针用于筛选副球菌种菌株R114的λ文库(见实施例4)。分离出了几个与phaA和phaC探针杂交的λ克隆,通过序列分析证实phaA和phaC基因是否存在于插入片段中。通过亚克隆和测序,进一步分析phaAλ-克隆,而在phaA基因的下游发现了phaB。因此,如同P.denitrificans的情形,phaA和phaB成簇在一起而phaC基因位于基因组中其它的位置。副球菌种菌株R114 phaAB簇的核苷酸序列和推定的乙酰-CoA乙酰基转移酶(PhaA)的氨基酸序列分别如SEQ ID NO177,SEQ ID NO178和179所示。PHA生物合成操纵子中相关基因成簇说明当细胞生成聚(3-烷羧酸酯)时,至少phaA和phaB是同时被表达的。另一方面,在副球菌种菌株R114中,在phaA和phaB基因之间发现了一个假定的转录停止信号,而这在P.denitrificans phaAB操纵子中是不存在的(Yabutani等,同上)。这样,这两个基因的表达在副球菌种菌株R114中可能不是相随的。
使用实施例5所述的常规克隆策略,phaA基因被克隆到质粒pDS-His中。将新的质粒——pDS-His-phaA——导入大肠杆菌M15。在具有IPTG诱导(如实施例5所述)和不具有IPTG诱导的条件下,培养获得的菌株M15/pDS-His-phaA,并制备粗提物用于SDS-PAGE分析。在IPTG诱导后,在大肠杆菌M15宿主中过量形成了克隆的His标记的副球菌种菌株R114PhaA。
以上已经提及了扩增atoB或phaA基因(编码乙酰-CoA乙酰基转移酶)在玉米黄质产量上的潜在优势。另外,降低或消除乙酰乙酰-CoA还原酶(phaB基因产物)的活性以避免一些在体内形成的乙酰乙酰-CoA转向PHA途径,也可以利于提高玉米黄质产量。可通过基因取代技术(特别是用无活性形式phaB基因取代野生型基因组中的phaB基因)或经典诱变和筛选技术,制备得到缺乏phaB活性的一些副球菌种菌株R114突变株。
实施例11使用副球菌种菌株R114衍生的突变株,工业生产番茄红素的范例番茄红素是一种红色的类胡萝卜素,是由菌株R-1512及其突变株R1534和R114所代表的新副球菌种中,玉米黄质生物合成中的中间体。由于番茄红素本身是一种重要的商品,所以,十分有必要测试新副球菌种通过工业发酵生产番茄红素的潜能。为获得玉米黄质生物合成被阻断的、能够积累番茄红素的突变株,用紫外线(UV)对副球菌种菌株R114进行诱变,然后筛选红色菌落。紫外诱变按下述进行在ME培养基(见实施例2)中过夜培养菌株R114。将该过夜培养物再次培养在新鲜的ME培养基中(起始OD610=0.1),并在28℃温育3小时。离心该培养物,用20mM磷酸钾溶液(pH7.2)洗涤沉淀。再次离心后,重悬浮沉淀,使终OD610为0.1。将4ml细胞悬浮液置于灭菌的100ml玻璃烧杯中。以1450μW/cm2的通量对这一薄层细胞悬浮液进行预先测定最佳时间长度的UV照射。在照射中,通过烧杯内的曲别针和磁力搅拌器,混合该细胞悬浮液。将诱变后的细胞悬浮液(和未突变的对照)培养在362/F2琼脂培养基上(表28)。在诱变前和诱变后,在悬浮液上进行三次存活平板计数。然后将该板在28℃孵育4-5天,为菌落评分。通过再-划线(re-streaking)鉴定及纯化几个红色菌落(假定的产番茄红素菌落)。一个突变株,命名为UV7-1,用于进一步评价番茄红素产量。
表29显示了对照菌株R114及其突变株UV7-1的玉米黄质产量和番茄红素产量。菌株R114只生成玉米黄质。突变株UV7-1生产大量的番茄红素,只生成少量玉米黄质,说明在UV7-1中(假设在crtE基因中)的突变阻断并不完全。这些结果说明可以从副球菌种菌株R114衍生出产番茄红素的菌株。
表28.培养基362F/2的配方和制备
表29.副球菌种菌株R114及其突变株UV7-1的玉米黄质产量和番茄红素产量
实施例12使用副球菌种菌株R114衍生的突变株,工业生产虾青素的范例虾青素是商业中一种重要的类胡萝卜素,主要用于水产养殖。EP0872,552说明通过导入含有副球菌种菌株R1534和Paracoccuscarotinifaciens E-396T克隆类胡萝卜素(crt)基因组合的质粒,可在大肠杆菌中形成虾青素。同时,克隆的crt基因(crtEBIYZ)和crtW(β-胡萝卜素β-4加氧酶)编码了从FPP通过玉米黄质生成虾青素的整个生物合成途径。P.carotinifaciens E-396 crtW,副球菌种R1534 crtZ和副球菌种R1534 crtE基因及编码多肽如(SEQ ID NO180和181(crtW);182和184(crtZ);及184和185(crtW))所示。但是,在副球菌种菌株R114宿主科(family)内未表现出可获得虾青素产量。为说明利用源自菌株R114的重组菌株进行虾青素合成,按下述将克隆的crtW基因(SEQ ID NO180)导入菌株R114中。
表30.实施例12中用于本文所述的PCR引物
使用引物crtW-Nde和crtW-Bam(表30),通过PCR,从Paracoccuscarotinifaciens菌株E-396T(Tsubokura等,同上,EP 872,554)的克隆crt簇扩增crtW基因。引物的设计使得ATG起始密码子构成了NdeI位点的后半部分(second half)(酶切识别位点CATATG),而在终止密码子后随即导入BamHI位点(GGATCC)。PCR产物被克隆到pCR_2.1-TOPO载体中,得到质粒TOPO-crtW。用NdeI和BamHI酶切crtW基因,并亚克隆到NdeI-BamHI酶切过的载体pBBR-K-PcrtE(实施例6所述)中,得到pBBR-K-PcrtE-crtW。
使用标准接合方法{大肠杆菌菌株S17[Priefer等,J.Bacteriol.163324-330(1985)]为供体有机体},将质粒pBBR-K-PcrtE-crtW转移到副球菌种菌株R114中。在含有50mg/l卡那霉素的362F/2琼脂培养基上选择转接合子,并在相同培养基上通过划线将其纯化。PCR验证质粒pBBR-K-PcrtE-crtW在菌株中的存在。除了使用362F/2培养基替代ME培养基之外,其余如实施例1和2所述,测定摇瓶培养物中菌株R114,R114/pBBR-K(空载体对照)和R114/pBBR-K-PcrtE-crtW的类胡萝卜素产量。结果如表31所示。对照菌株R114和R114/pBBR-K仅生成玉米黄质。在菌株R114/pBBR-K-PcrtE-crtW中,玉米黄质完全被质粒编码的β-胡萝卜素β-4加氧酶消耗。尽管生成了虾青素,但是,其它两种酮类胡萝卜素(ketocarotenoid),福寿草黄素(adonixanthin)和角黄素积累的水平很高。这说明体内β-胡萝卜素羟化酶(菌株R114中由染色体crtZ基因编码)和克隆的β-胡萝卜素β-4加氧酶(CrtW)不平衡。
为证实该假设,制备两种新的质粒,其在Mini操纵子中同时含有crtZ和crtW基因。使两种构建体中基因的顺序不相同(即crtZ-crtW和crtW-crtZ),以试图形成不同的crtZ和crtW基因表达比率。新载体的构建需要按下述组装特定的克隆载体组。设计一系列操纵子构建载体(根据载体pCR_2.1-TOPO),以促进将基因(在此crtZ和crtW)组装到操纵子中。目的基因必须具有ATG起始密码子和TGA终止密码子,起始密码子应位于NdeI位点中(CATATG),终止密码子应直接位于BamHI位点之后。
表31.含有表达单独crtW基因、以及crtW和crtZ基因组合的质粒的副球菌种菌株R114中虾青素产量
aZXN,玉米黄质;AND福寿草黄素(adonixanthin);CXN角黄素;AXN虾青素;b比形成,表达为mg/l总类胡萝卜素/OD660另外,起始密码子后的第一个核苷酸和终止密码子之前的最后一个核苷酸必须是腺嘌呤,该基因必须缺乏至少一个下述酶的位点BsgI,BseMII,BseRI和GsuI。构建四个操纵子构建载体,其区别在于其多接头序列(SEQ IDNO190-197)的排列。前两个酶的分裂位点在NdeI位点中。后两个酶的分裂位点在BamHI位点之前。pOCV-1和pOCV-4中的BseRI位点并不唯一,且不能用于构建操纵子。
首先,将待组装入操纵子的各基因单独地插入到适当操纵子构建载体中的NdeI和BamHI位点之间。然后,将带有想象操纵子上游基因的所获质粒用多接头末尾两个酶之一和一种在载体骨架中具有独特位点的酶进行酶切。用多接头末尾两个酶之一和用于第一个质粒(含有期望的上游基因)相同的酶(在载体骨架中具有独特位点),酶切含有想象操纵子下游基因的质粒。分离出带有各基因的片段,并连接,获得在NdeI和BamHI位点之间带有这两个基因的pOCV质粒。可以类似的方式加入更多的基因。组装的各基因相互重叠,使得上游基因TGA终止密码子的前两个核苷酸TG与下游基因起始密码子ATG的后两个核苷酸重合。在副球菌种菌株R1534类胡萝卜素(crt)操纵子(crtZYIB)的所有基因之间均发现了这种重叠(Pasamontes等,同上)。
pOCV骨架来自pCR_2.1-TOPO。通过定点突变,将位点CTCAG改变为CACAG,消除了复制必须区域,ColE1起始位点上游中的BseMII位点。通过去除一段0.8kb的DdeI-Asp700片段,消除剩余的三个BseMII位点和一个GsuI位点。填平DdeI的凹缺末端后,该载体变为平头末端连接。通过使寡核苷酸与适当的5’突出端一起退火,在BamHI和XbaI位点之间插入多接头。
使用操纵子构建载体pOCV-2,按下述构建质粒pBBR-K-PcrtE-crtZW。使用引物crtZ-Nde和crtZ-Bam(表30),通过PCR,从副球菌种菌株R114扩增crtZ基因。引物的设计使得ATG起始密码子构成了NdeI位点的后半部分(酶切识别位点CATATG),而在终止密码子后随即导入BamHI位点(GGATCC)。PCR产物被克隆到pCR_2.1-TOPO载体中,得到质粒TOPO-crtZ。为将两个基因组装到一个Mini操纵子中,用NdeI和BamHI酶切质粒TOPO-crtZ和TOP-crtW的crtZ和crtW,并亚克隆到NdeI-BamHI酶切过的载体pOCV-2中,得到质粒pOCV-2-crtZ和pOCV-2-crtW。用BseRI和PstI(在卡那霉素抗性基因中存在一个独特的PstI位点)酶切质粒pOCV-2-crtZ,将2.4kb片段(含有crtZ)与来自pOCV-2-crtW的含crtW的1876bp BseRI-PstI片段相连。所获的质粒pOCV-2-crtZW用NdeI和BamHI酶切,并与pBBR-K-PcrtE的NdeI-BamHI骨架相连,得到pBBR-K-PcrtE-crtZW。以类似的方式构建质粒pBBR-K-PcrtE-crtWZ。
表31中的数据表明与菌株pBBR-K-PcrtE-crtW相比,菌株R114/pBBR-K-PcrtE-crtWZ中的福寿草黄素(adonixanthin),角黄素和虾青素比率并没有显著地改变。但是,在pBBR-K-PcrtE-crtZW中,酮类胡萝卜素(ketocarotenoid)的产量转变为以虾青素为主。该结果说明表达水平依赖于基因在Mini操纵子中的位置,并暗示着增强体内β-胡萝卜素羟化酶的活性水平可以提高该酶和β-胡萝卜素β-4加氧酶活性之间的平衡,而β-胡萝卜素β-4加氧酶对于将玉米黄质完全转化为虾青素更为有利。
该实施例所述的结果还显示通过适当的遗传操作,不仅可以在副球菌种菌株R114或其亲缘菌株内产生虾青素,还可以生成其它目的酮类胡萝卜素商品。例如,在菌株R114的crtZ突变株(缺乏β-胡萝卜素羟化酶活性)中表达编码β-胡萝卜素β-4加氧酶的基因,可以专一性地生产酮类胡萝卜素,如,海胆酮或角黄素,而不会同时生成羟基化的类胡萝卜素。综上,该实施例和实施例11的结果说明副球菌种菌株R114及其亲缘菌株在工业化生产重要类胡萝卜素中具有光明的应用前景。
实施例13在培养过量表达甲羟戊酸途径各基因的副球菌种菌株R114过程中甲羟戊酸的积累在副球菌种菌株R114中过量表达甲羟戊酸途径的各基因,可以增强碳流动通过甲羟戊酸途径。实施例5描述了质粒pBBR-K-mev-op16-2的构建。按下述构建质粒pBBR-K-mev-op-up-4。对含有副球菌种菌株R114甲羟戊酸操纵子的λ克隆进行部分酶切后(参见实施例4),在3.1kb SmaI-SalI片段上获得了含有mvaA基因绝大部分以及整个idi和hcs基因的DNA片段。将该片段亚克隆到pUC19中,得到质粒pUC19mev-op-up’。为促进亚克隆,将含有甲羟戊酸各基因的pUC19mev-op-up’的KpnI-HindIII片段再克隆到载体pBluescriptKS+中,得到质粒pBluKSp-mev-op-up′。然后将来自pUC19mev-op-up’的一段1.7kb SalI片段克隆到质粒2ES2-1的SalI位点,该质粒为pUC19衍生的质粒,含有副球菌种菌株R114的克隆SalI-EcoRI片段M(参见实施例4)。这样形成了质粒pUC 19mev-op-up-2。然后将pUC19mev-op-up-2中带有甲羟戊酸操纵子起始部分的BbsI-BsaI片段与pBluKSp-mev-op-up’中含有idi和hcs的BbsI-BsaI片段相结合,得到质粒pUC19mev-op-up-3。另外,通过插入一段含有MluI限制性位点的退火引物,将单一的MluI位点导入载体pBBR-1MCS-2(见实施例5)的NsiI和KpnI位点。用MluI和KpnI酶切新获得的克隆载体pBBR-K-Mlu,并将pUCmev-op-up-3的MluI-KpnI片段(该片段含有甲羟戊酸操纵子的前三个基因)插入,获得质粒pBBR-K-mev-op-up-3。然后,插入质粒16SB3的SmaI片段,构建质粒pBBR-K-mev-op-up-4,所述16SB3含有mvk基因的绝大部分和pmk的5′末端(质粒16SB3是衍生自pUC19的质粒,含有副球菌种菌株R114 SalI-BamHI片段A;参见实施例4)。pBBR-K-mev-op-up-4的插入部分含有推定的甲羟戊酸操纵子启动子区域,甲羟戊酸操纵子的前四个基因和pmk的5′末端。
通过电穿孔,将质粒pBBR-K-mev-op16-2和pBBR-K-mev-op-up-4分别导入副球菌种菌株R114。对比新菌株与对照菌株R114的玉米黄质和甲羟戊酸产量。在带挡板摇瓶中,用液体培养基362F/2(见实施例11)培养各菌株72小时。对于菌株R114/pBBR-K-mev-op16-2和菌株R114/pBBR-K-mev-op-up-4,还向培养基中加入卡那霉素(50mg/l)。培养温度为28℃,摇瓶速度为200rpm。通过实施例1所述方法测定玉米黄质,而培养物上清中甲羟戊酸的测定按下述进行将0.6ml培养物样品13000xg离心4分钟。将400微升上清加入到400微升甲醇中,并涡旋搅拌1min。再次将混合物以13000xg离心4分钟。然后,使用Lindemann的方法[J.Pharm.Biomed.Anal.9311-316(1991)],将所获上清直接用气相色谱法(GC)进行分析,但其中几处微小变动如下所述GC是Hewlett-Packard 6890+仪器(Hewlett-Packard,Avondale,PA,USA),其配有柱上冷却(cool-on-column)注射器和火焰离子化检测器。将1微升如上制备的样品注入熔融石英毛细管柱(15m长×0.32mm ID)中,所述柱子已用0.52微米交联修饰聚乙二醇(HP-FFAP,Agilent Technologies,USA)薄膜覆盖。进口压力0.6巴,以氦气作为载体气体。可程序化注射器的温度以30℃/分钟的速率从82℃向250℃上升。柱子温度曲线为80℃0.5分钟,然后以15℃/分钟的线性温度梯度升到250℃,最后在250℃维持5分钟。检测温度维持在320℃。
在第一个试验中,测定菌株R114和R114/pBBR-K-mev-op16-2的玉米黄质和甲羟戊酸产量(表32)。两个菌株的玉米黄质产量类似,但是菌株R114/pBBR-K-mev-op16-2的甲羟戊酸产量水平要高出4倍。这些结果显示在副球菌种菌株R114中过量表达甲羟戊酸途径各基因可以提高碳流动通过甲羟戊酸途径。预先估计到了甲羟戊酸会积累,因为菌株R114/pBBR-K-mev-op16-2不具有过量表达的crtE基因,而已知crtE基因的产物(GGPP合成酶)是副球菌种菌株R114中玉米黄质生成的限速步骤(见实施例6和8)。可以预期具有有限数量GGPP合成酶的细胞在过量生成甲羟戊酸途径各酶之后,会积累FPP,而众所周知FPP是甲羟戊酸即酶的有效抑制剂[Dorsey和Porter,J.Biol.Chem.2434667-4670(1968);Gray和Kekwick,BBA 279290-296(1972);Hinson等,J.Lipids Res.382216-2223(1997)]。因此,由于过量表达甲羟戊酸途径各基因而引起的FPP的积累,将会导致对甲羟戊酸激酶的抑制作用,而这反过来可以表明培养过程中甲羟戊酸的积累。
表32.菌株R114和R114/pBBR-K-mev-op16-2的玉米黄质和甲羟戊酸产量
在第二个试验中,测定菌株R114和R114/pBBR-K-mev-op-up-4的两个独立分离株的玉米黄质和甲羟戊酸产量(表33)。这些结果再次表明过量表达甲羟戊酸途径各基因可以提高碳流动通过甲羟戊酸途径。
表33.菌株R114和R114/pBBR-K-mev-op-up-4的玉米黄质和甲羟戊酸产量
根据布达佩斯条约,下述生物材料保藏在美国典型培养物保藏中心(American Type Culture Collection(ATCC)),0801 University Blvd.,ManassasVA 20110-2201,USA,登录号如下
以上引用的所有专利,专利申请和出版物均将其全部引入本文作为参考,如同将每篇在本文中全文引用一样。
本文所公开的发明,很明显可以通过各种方式对其进行修改。这种改变仍然属于本发明的精神和范围之内,这样的修改仍落入下面的权利要求范围中。
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<221>misc_feature<222>(24)..(24)<223>n代表C或G<220>
<221>misc_feature<222>(1)..(24)<223>寡核苷酸<400>32acnatggang cnggnccnaa ngtn 24<210>33<211>24<212>DNA<213>人工的<220>
<221>misc_feature<222>(3)..(3)<223>n代表C或G<220>
<221>misc_feature<222>(9)..(9)<223>n代表A或G<220>
<221>misc_feature<222>(12)..(12)<223>n代表A或G<220>
<221>misc_feature<222>(12)..(12)<223>n代表C或G<220>
<221>misc_feature<222>(15)..(15)<223>n代表A或C或G<220>
<221>misc_feature<222>(18)..(18)<223>n代表C或G<220>
<221>misc_feature<222>(21)..(21)<223>n代表A或G<220>
<221>misc_feature<222>(24)..(24)<223>n代表C或G<220>
<221>misc_feature<222>(1)..(24)<223>寡核苷酸<400>33tgntacctnc gnccnggntt ncan 24
<210>34<211>17<212>DNA<213>人工的<220>
<221>misc_feature<222>(12)..(12)<223>n代表C或G<220>
<221>misc_feature<222>(15)..(15)<223>n代表A或C或G<220>
<221>misc_feature<222>(1)..(17)<223>寡核苷酸<400>34tggtacctac gnccngg 17<210>35<211>17<212>DNA<213>人工的<220>
<221>misc_feature<222>(12)..(12)<223>n代表C或G<220>
<221>misc_feature<222>(15)..(15)<223>n代表A或C或G<220>
<221>misc_feature<222>(1)..(17)<223>寡核苷酸<400>35tggtacctgc gnccngg17<210>36<211>17<212>DNA<213>人工的<220>
<221>misc_feature<222>(12)..(12)<223>n代表C或G<220>
<221>misc_feature<222>(1)..(17)<223>寡核苷酸<220>
<221>misc_feature<222>(15)..(15)<223>n代表A或C或G<400>36tgctacctac gnccngg 17
<210>37<211>17<212>DNA<213>人工的<220>
<221>misc_feature<222>(12)..(12)<223>n代表C或G<220>
<221>misc_feature<222>(15)..(15)<223>n代表A或C或G<220>
<221>misc_feature<222>(1)..(17)<223>寡核苷酸<400>37tgctacctgc gnccngg 17<210>38<211>20<212>DNA<213>人工的<220>
<221>misc_feature<222>(9)..(9)<223>n代表C或G<220>
<221>misc_feature<222>(12)..(12)<223>n代表A或C或G<220>
<221>misc_feature<222>(15)..(15)<223>n代表C或G<220>
<221>misc_feature<222>(18)..(18)<223>n代表A或G<220>
<221>misc_feature<222>(1)..(20)<223>寡核苷酸<400>38tacctacgnc cnggnttnca 20<210>39<211>20<212>DNA<213>人工的<220>
<221>misc_feature<222>(9)..(9)<223>n代表C或G<220>
<221>misc_feature<222>(12)..(12)<223>n代表A或C或G
<220>
<221>misc_feature<222>(15)..(15)<223>n代表C或G<220>
<221>misc_feature<222>(18)..(18)<223>n代表A或G<220>
<221>misc_feature<222>(1)..(20)<223>寡核苷酸<400>39tacctgcgnc cnggnttnca 20<210>40<211>20<212>DNA<213>人工的<220>
<221>misc_feature<222>(9)..(9)<223>n代表C或G<220>
<221>misc_feature<222>(12)..(12)<223>n代表A或C或G<220>
<221>misc_feature<222>(15)..(15)<223>n代表C或G<220>
<221>misc_feature<222>(18)..(18)<223>n代表C或T<220>
<221>misc_feature<222>(1)..(20)<223>寡核苷酸<400>40tacctacgnc cnggngtnca 20<210>41<211>20<212>DNA<213>人工的<220>
<221>misc_feature<222>(9)..(9)<223>n代表C或G<220>
<221>misc_feature<222>(12)..(12)<223>n代表A或C或G<220>
<221>misc_feature<222>(15)..(15)<223>n代表C或G<220>
<221>misc_feature
<222>(18)..(18)<223>n代表C或T<220>
<221>misc_feature<222>(1)..(20)<223>寡核苷酸<400>41tacctgcgnc cnggngtnca 20<210>42<211>9066<212>DNA<213>副球菌属菌种(Paracoccus sp.)<220>
<221>CDS<222>(2622)..(3644)<223>
<400>42ggatccggca gctcgacacg ccgcagaacc tgtacgaacg tcccgccagc cgcttcgtcg 60cggaattcgt cgggcgcggg acggtggtgc ccgtgcaggc ccatgacggc gcgggccgcg120cccgcatcct gggggccgag gtggcggtga acgccgcccc gcaatcgcgc tttgtcgatc180acgtctgcct gcgccccgag aaccttgcca tctccgagac gggcgacctg cgcgccaagg240tcgcgcgcgt cacctatctt ggcgggaaat acctgctgga aaccgtgctg gattgcggca300cccggctggt gaccgagacc cgcgcccgct tcgatacggg cgcgcagctt ggcctgacca360tcaacgcccc ctgggccttt gccgaggatt gaatggacag cgtgaagatc ctttcgggca420tgggcgtgaa gggccctgcc tgcatcaggc tggatgtcgg cgggatgcgc ctgatcctcg480attgcgggac cggcccggac gagggcgcgg agttcgaccc cgcctggctg gcggacgcgg540atgcggtgct gatcacccat gaccacgtgg accatatcgg cggcgcgcgt cacgcggtcg600cggcggggct gccgatccat gcgacgcggc agacggcggg gttgctgccc gcgggggcgg660atctgcgcct gctgcccgaa cgcggtgtca cgcggatcgc cggggtcgat ctgacgaccg720gtcgcaacgg gcatgccgcg ggcggcgtct ggatgcattt cgacatgggc gaggggctgt780tctattccgg cgactggtcc gaggaatccg actggttcgc cttcgatccg cccccgcctg840cggggacggc gattctcgac tgctcctatg gcggtttcga cgtggcgcaa tcggattgca900tcgcggacct ggacgacctg ctcgaggtgc tgccggggca ggtactgctg ccggtgccgc960catccggccg cgcggccgag ctggccctgc ggctgatccg ccgccacgga ccgggcagcg 1020tgatggtcga cgacgcctgc ctgccggcca tcgcgcaact gcccgaggcg cgcggactgg 1080cctacgccac cgaggcacgc tttcttgtct gcgacacgcc gaacgccgaa agccggcgcg 1140gcatggcggc atctgcaagc atggcgcgat gcgggcaggc tggggcggga cgcgcatgtc 1200gtcttcaccg ggcacatgaa cgtccatgcg cgcgcattct gcgaccgccc cggcgggcat 1260ttccgccgct ggaacgtgca tccgccgctg cgcgaccagc gacggatgct ggaacggctg 1320gccgcgcggc gctttgcccc ggccttctgc cccgaccccg agatctatct ggcgctggac 1380atgggcgcgc aggtcttcat gcaccaggag gtgacgccat gatccccgcc cgcagcttct 1440gcctgatccg ccacggcgaa acgaccgcca atgcaggggc gatcatcgcg ggcgcaaccg 1500atgtgcccct gacgccaagg ggccgcgatc aggcccgcgc cctggcaggg cgcgaatggc 1560catcgggcat cgcgctgttc gccagcccga tgtcgcgtgc ccgcgatacc gcgctgctgg 1620cctttccggg gcgcgaccac cagcccgaac ccgatctgcg cgaacgcgac tggggcatct 1680tcgagggacg ccccgtcgcc gatctgcccc cgcgcgaaat cacgccgcag gggggcgagg 1740gctgggacga cgtgatggcc cgcgtggacc gcgcgatccg gcggatctgc gcgacctcgg 1800gcgatgcgct gccggtgctg gtctgccatt cgggcgtgat ccgtgccgcg cgcgtgctgt 1860ggaccaccgg cgatgcgggc gatcgtccgc ccaacgccac gccgatcctg ttcagcccgg 1920acggcgaccg attaaaggaa ggaacgatat gaccgccacc accccctgcg tcgtcttcga 1980acgtggacgg cacgcttgcc gaattcgacg ccgaccgcct gggccatctt gtccacggca 2040cgaccaagca ctgggacgcc ttccaccacg cgatggccga cgccccgccc atccccgagg 2100tcgcccgcct gatgcgcaag ctgaaggagg ggggcgagac ggtcgtcatc tgctcggggc 2160ggccccgcgg ctggcaggat cagacgatcg catggctgcg caagcacgac ctgcccttcg 2220acgggatcta tctgcgcccc gaggatcagg acggcgccag cgaccccgag gtcaagcgcc 2280gcgccctagc cgagatgcgc gccgacgggc tggcgccctg gctggtcgtg gacgaccggc 2340ggtccgtcgt ggatgcctgg cgggccgagg ggctggtctg cctgcaatgc gcgccggggg 2400acttctaggg ccgcgcgacg ggggcgcgga caggctgggc gggaaaccgc cccgccacca 2460tgtcctgcac gcgtcgaacc gcccgtccga cgccggtttc cgcacggaaa cgcgcggcaa 2520
gttgacataa cttgcacgcg acgtctcgat tctgcccgcg aagaatgcga tgcatccaga2580tgatgcagaa cgaagaagcg gaagcgcccg tgaaagacca g atg att tcc cat acc2636Met Ile Ser His Thr1 5ccg gtg ccc acg caa tgg gtc ggc ccg atc ctg ttc cgc ggc ccc gtc 2684Pro Val Pro Thr Gln Trp Val Gly Pro Ile Leu Phe Arg Gly Pro Val10 15 20gtc gag ggc ccg atc agc gcg ccg ctg gcc acc tac gag acg ccg ctc 2732Val Glu Gly Pro Ile Ser Ala Pro Leu Ala Thr Tyr Glu Thr Pro Leu25 30 35tgg ccc tcg acc gcg cgg ggg gca ggg gtt tcc cgg cat tcg ggc ggg 2780Trp Pro Ser Thr Ala Arg Gly Ala Gly Val Ser Arg His Ser Gly Gly40 45 50atc cag gtc tcg ctg gtc gac gaa cgc atg agc cgc tcg atc gcg ctg 2828Ile Gln Val Ser Leu Val Asp Glu Arg Met Ser Arg Ser Ile Ala Leu55 60 65cgg gcg cat gac ggg gcg gcg gcg acc gcc gcc tgg cag tcg atc aag 2876Arg Ala His Asp Gly Ala Ala Ala Thr Ala Ala Trp Gln Ser Ile Lys70 75 80 85gcc cgc cag gaa gag gtc gcg gcc gtg gtc gcc acc acc agc cgc ttc 2924Ala Arg Gln Glu Glu Val Ala Ala Val Val Ala Thr Thr Ser Arg Phe90 95 100gcc cgc ctt gtc gag ctg aat cgc cag atc gtg ggc aac ctg ctt tac 2972Ala Arg Leu Val Glu Leu Asn Arg Gln Ile Val Gly Asn Leu Leu Tyr105 110 115atc cgc atc gaa tgc gtg acg ggc gac gcc tcg ggt cac aac atg gtc 3020Ile Arg Ile Glu Cys Val Thr Gly Asp Ala Ser Gly His Asn Met Val120 125 130acc aag gcc gcc gag gcc gtg cag ggc tgg atc ctg tcg gaa tac ccg 3068Thr Lys Ala Ala Glu Ala Val Gln Gly Trp Ile Leu Ser Glu Tyr Pro135 140 145atg ctg gcc tat tcc acg atc tcg ggg aac ctg tgc acc gac aag aag 3116Met Leu Ala Tyr Ser Thr Ile Ser Gly Asn Leu Cys Thr Asp Lys Lys150 155 160 165gcg tcg gcg gtc aac ggc atc ctg ggc cgc ggc aaa tac gcc gtc gcc 3164Ala Ser Ala Val Asn Gly Ile Leu Gly Arg Gly Lys Tyr Ala Val Ala170 175 180gag gtc gag atc ccg cgc aag atc ctg acc cgc gtg ctg cgc acc agc 3212Glu Val Glu Ile Pro Arg Lys Ile Leu Thr Arg Val Leu Arg Thr Ser185 190 195gcc gag aag atg gtc cgc ctg aac tac gag aag aac tat gtc ggg ggt 3260Ala Glu Lys Met Val Arg Leu Asn Tyr Glu Lys Asn Tyr Val Gly Gly200 205 210acg ctg gcg ggg tcg ctg cgc agt gcg aac gcg cat ttc gcc aac atg 3308Thr Leu Ala Gly Ser Leu Arg Ser Ala Asn Ala His Phe Ala Asn Met215 220 225
ctg ctg ggc ttc tac ctg gcg acg ggg cag gac gcg gcc aac atc atc 3356Leu Leu Gly Phe Tyr Leu Ala Thr Gly Gln Asp Ala Ala Asn Ile Ile230 235 240 245gag gcc agc cag ggc ttc gtc cat tgc gag gcc cgc ggc gag gat ctg 3404Glu Ala Ser Gln Gly Phe Val His Cys Glu Ala Arg Gly Glu Asp Leu250 255 260tat ttc tcg tgc acg ctg ccc aac ctc atc atg ggc tcg gtc ggt gcc 3452Tyr Phe Ser Cys Thr Leu Pro Asn Leu Ile Met Gly Ser Val Gly Ala265 270 275ggc aag ggc atc ccc tcg atc gag gag aac ctg tcg cgg atg ggc tgc 3500Gly Lys Gly Ile Pro Ser Ile Glu Glu Asn Leu Ser Arg Met Gly Cys280 285 290cgc cag ccg ggc gaa ccc ggc gac aac gcg cgc cgt ctt gcg gcg atc 3548Arg Gln Pro Gly Glu Pro Gly Asp Asn Ala Arg Arg Leu Ala Ala Ile295 300 305tgc gcg ggc gtc gtg ctg tgt ggt gaa ttg tcg ctg ctt gcg gcc cag 3596Cys Ala Gly Val Val Leu Cys Gly Glu Leu Ser Leu Leu Ala Ala G1n310 315 320 325acc aac ccc gga gag ttg gtc cgc acc cac atg gag atg gag cga tga 3644Thr Asn Pro Gly Glu Leu Val Arg Thr His Met Glu Met Glu Arg330 335 340ccgacagcaa ggatcaccat gtcgcggggc gcaagctgga ccatctgcgt gcattggacg3704acgatgcgga tatcgaccgg ggcgacagcg gcttcgaccg catcgcgctg acccatcgcg3764ccctgcccga ggtggatttc gacgccatcg acacggcgac cagcttcctg ggccgtgaac3824tgtccttccc gctgctgatc tcgtccatga ccggcggcac cggcgaggag atcgagcgca3884tcaaccgcaa cctggccgct ggtgccgagg aggcccgcgt cgccatggcg gtgggctcgc3944agcgcgtgat gttcaccgac ccctcggcgc gggccagctt cgacctgcgc gcccatgcgc4004ccaccgtgcc gctgctggcc aatatcggcg cggtgcagct gaacatgggg ctggggctga4064aggaatgcct ggccgcgatc gaggtgctgc aggcggacgg cctgtatctg cacctgaacc4124ccctgcaaga ggccgtccag cccgaggggg atcgcgactt tgccgatctg ggcagcaaga4184tcgcggccat cgcccgcgac gttcccgtgc ccgtcctgct gaaggaggtg ggctgcggcc4244tgtcggcggc cgatatcgcc atcgggctgc gcgccgggat ccggcatttc gacgtggccg4304gtcgcggcgg cacatcctgg agccggatcg agtatcgccg ccgccagcgg gccgatgacg4364acctgggcct ggtcttccag gactggggcc tgcagaccgt ggacgccctg cgcgaggcgc4424ggcccgcgct tgcggcccat gatggaacca gcgtgctgat cgccagcggc ggcatccgca4484acggtgtcga catggcgaaa tgcgtcatcc tgggggccga catgtgcggg gtcgccgcgc4544ccctgctgaa agcggcccaa aactcgcgcg aggcggttgt atccgccatc cggaaactgc4604atctggagtt ccggacagcc atgttcctcc tgggttgcgg cacgcttgcc gacctgaagg4664acaattcctc gcttatccgt caatgaaagt gcctaagatg accgtgacag gaatcgaagc4724gatcagcttc tacacccccc agaactacgt gggactggat atccttgccg cgcatcacgg4784gatcgacccc gagaagttct cgaaggggat cgggcaggag aaaatcgcac tgcccggcca4844tgacgaggat atcgtgacca tggccgccga ggccgcgctg ccgatcatcg aacgcgcggg4904cacgcagggc atcgacacgg ttctgttcgc caccgagagc gggatcgacc agtcgaaggc4964cgccgccatc tatctgcgcc gcctgctgga cctgtcgccc aactgccgtt gcgtcgagct5024gaagcaggcc tgctattccg cgacggcggc gctgcagatg gcctgcgcgc atgtcgcccg5084caagcccgac cgcaaggtgc tggtgatcgc gtccgatgtc gcgcgctatg accgcgaaag5144ctcgggcgag gcgacgcagg gtgcgggcgc cgtcgccatc cttgtcagcg ccgatcccaa5204ggtggccgag atcggcaccg tctcggggct gttcaccgag gatatcatgg atttctggcg5264gccgaaccac cgccgcacgc ccctgttcga cggcaaggca tcgacgctgc gctatctgaa5324cgcgctggtc gaggcgtgga acgactatcg cgcgaatggc ggccacgagt tcgccgattt5384cgcgcatttc tgctatcacg tgccgttctc gcggatgggc gagaaggcga acagccacct5444ggccaaggcg aacaagacgc cggtggacat ggggcaggtg cagacgggcc tgatctacaa5504ccggcaggtc gggaactgct ataccgggtc gatctacctg gcattcgcct cgctgctgga5564gaacgctcag gaggacctga ccggcgcgct ggtcggtctg ttcagctatg gctcgggtgc5624
gacgggcgaa ttcttcgatg cgcggatcgc gcccggttac cgcgaccacc tgttcgcgga5684acgccatcgc gaattgctgc aggatcgcac gcccgtcaca tatgacgaat acgttgccct5744gtgggacgag atcgacctga cgcagggcgc gcccgacaag gcgcgcggtc gtttcaggct5804ggcaggtatc gaggacgaga agcgcatcta tgtcgaccgg caggcctgaa gcaggcgccc5864atgccccggg caagctgatc ctgtccgggg aacattccgt gctctatggt gcgcccgcgc5924ttgccatggc catcgcccgc tataccgagg tgtggttcac gccgcttggc attggcgagg5984ggatacgcac gacattcgcc aatctctcgg gcggggcgac ctattcgctg aagctgctgt6044cggggttcaa gtcgcggctg gaccgccggt tcgagcagtt cctgaacggc gacctaaagg6104tgcacaaggt cctgacccat cccgacgatc tggcggtcta tgcgctggcg tcgcttctgc6164acgacaagcc gccggggacc gccgcgatgc cgggcatcgg cgcgatgcac cacctgccgc6224gaccgggtga gctgggcagc cggacggagc tgcccatcgg cgcgggcatg gggtcgtctg6284cggccatcgt cgcggccacc acggtcctgt tcgagacgct gctggaccgg cccaagacgc6344ccgaacagcg cttcgaccgc gtccgcttct gcgagcggtt gaagcacggc aaggccggtc6404ccatcgacgc ggccagcgtc gtgcgcggcg ggcttgtccg cgtgggcggg aacgggccgg6464gttcgatcag cagcttcgat ttgcccgagg atcacgacct tgtcgcggga cgcggctggt6524actgggtact gcacgggcgc cccgtcagcg ggaccggcga atgcgtcagc gcggtcgcgg6584cggcgcatgg tcgcgatgcg gcgctgtggg acgccttcgc agtctgcacc cgcgcgttgg6644aggccgcgct gctgtctggg ggcagccccg acgccgccat caccgagaac cagcgcctgc6704tggaacgcat cggcgtcgtg ccggcagcga cgcaggccct cgtggcccag atcgaggagg6764cgggtggcgc ggccaagatc tgcggcgcag gttccgtgcg gggcgatcac ggcggggcgg6824tcctcgtgcg gattgacgac gcgcaggcga tggcttcggt catggcgcgc catcccgacc6884tcgactgggc gcccctgcgc atgtcgcgca cgggggcggc acccggcccc gcgccgcgtg6944cgcaaccgct gccggggcag ggctgatgga tcaggtcatc cgcgccagcg cgccgggttc7004ggtcatgatc acgggcgaac atgccgtggt ctatggacac cgcgccatcg tcgccgggat7064cgagcagcgc gcccatgtga cgatcgtccc gcgtgccgac cgcatgtttc gcatcacctc7124gcagatcggg gcgccgcagc aggggtcgct ggacgatctg cctgcgggcg ggacctatcg7184cttcgtgctg gccgccatcg cgcgacacgc gccggacctg ccttgcgggt tcgacatgga7244catcacctcg gggatcgatc cgaggctcgg gcttggatcc tcggcggcgg tgacggtcgc7304ctgcctcggc gcgctgtcgc ggctggcggg gcgggggacc gaggggctgc atgacgacgc7364gctgcgcatc gtccgcgcca tccagggcag gggcagcggg gccgatctgg cggccagcct7424gcatggcggc ttcgtcgcct atcgcgcgcc cgatggcggt gccgcgcaga tcgaggcgct7484tccggtgccg ccggggccgt tcggcctgcg ctatgcgggc tacaagaccc cgacagccga7544ggtgctgcgc cttgtggccg atcggatggc gggcaacgag gccgctttcg acgcgctcta7604ctcccggatg ggcgcaagcg cagatgccgc gatccgcgcg gcgcaagggc tggactgggc7664tgcattccac gacgcgctga acgaatacca gcgcctgatg gagcagctgg gcgtgtccga7724cgacacgctg gacgcgatca tccgcgaggc gcgcgacgcg ggcgccgcag tcgccaagat7784ctccggctcg gggctggggg attgcgtgct ggcactgggc gaccagccca agggtttcgt7844gcccgcaagc attgccgaga agggacttgt tttcgatgac tgatgccgtc cgcgacatga7904tcgcccgtgc catggcgggc gcgaccgaca tccgagcagc cgaggcttat gcgcccagca7964acatcgcgct gtcgaaatac tggggcaagc gcgacgccgc gcggaacctt ccgctgaaca8024gctccgtctc gatctcgttg gcgaactggg gctctcatac gcgggtcgag gggtccggca8084cgggccacga cgaggtgcat cacaacggca cgctgctgga tccgggcgac gccttcgcgc8144gccgcgcgtt ggcattcgct gacctgttcc ggggggggag gcacctgccg ctgcggatca8204cgacgcagaa ctcgatcccg acggcggcgg ggcttgcctc gtcggcctcg gggttcgcgg8264cgctgacccg tgcgctggcg ggggcgttcg ggctggatct ggacgacacg gatctgagcc8324gcatcgcccg gatcggcagt ggcagcgccg cccgctcgat ctggcacggc ttcgtccgct8384ggaaccgggg cgaggccgag gatgggcatg acagccacgg cgtcccgctg gacctgcgct8444ggcccggctt ccgcatcgcg atcgtggccg tggacaaggg gcccaagcct ttcagttcgc8504gcgacggcat gaaccacacg gtcgagacca gcccgctgtt cccgccctgg cctgcgcagg8564cggaagcgga ttgccgcgtc atcgaggatg cgatcgccgc ccgcgacatg gccgccctgg8624gtccgcgggt cgaggcgaac gcccttgcga tgcacgccac gatgatggcc gcgcgcccgc8684cgctctgcta cctgacgggc ggcagctggc aggtgctgga acgcctgtgg caggcccgcg8744cggacgggct tgcggccttt gcgacgatgg atgccggccc gaacgtcaag ctgatcttcg8804aggaaagcag cgccgccgac gtgctgtacc tgttccccga cgccagcctg atcgcgccgt8864tcgaggggcg ttgaacgcgt aagacgacca ctgggtaagg ttctgccgcg cgtggtctcg8924actgcctgca aagaggtgct tgagttgctg cgtgactgcg gcggccgact tcgtgggact8984tgcccgccac gctgacgcgc tggaaacgcg cccgcggatt acgaccgcgt cattgccctg9044aaccaatttc ccgtcggtcg ac 9066
<210>43<211>340<212>PRT<213>副球菌属菌种(Paracoccus sp.)<400>43Met Ile Ser His Thr Pro Val Pro Thr Gln Trp Val Gly pro Ile Leu1 5 10 15Phe Arg Gly Pro Val Val Glu Gly Pro Ile Ser Ala Pro Leu Ala Thr20 25 30Tyr Glu Thr Pro Leu Trp Pro Ser Thr Ala Arg Gly Ala Gly Val Ser35 40 45Arg His Ser Gly Gly Ile Gln Val Ser Leu Val Asp Glu Arg Met Ser50 55 60Arg Ser Ile Ala Leu Arg Ala His Asp Gly Ala Ala Ala Thr Ala Ala65 70 75 80Trp Gln Ser Ile Lys Ala Arg Gln Glu Glu Val Ala Ala Val Val Ala85 90 95Thr Thr Ser Arg Phe Ala Arg Leu Val Glu Leu Asn Arg Gln Ile Val100 105 110Gly Asn Leu Leu Tyr Ile Arg Ile Glu Cys Val Thr Gly Asp Ala Ser115 120 125Gly His Asn Met Val Thr Lys Ala Ala Glu Ala Val Gln Gly Trp Ile130 135 140Leu Ser Glu Tyr Pro Met Leu Ala Tyr Ser Thr Ile Ser Gly Asn Leu145 150 155 160Cys Thr Asp Lys Lys Ala Ser Ala Val Asn Gly Ile Leu Gly Arg Gly165 170 175Lys Tyr Ala Val Ala Glu Val Glu Ile Pro Arg Lys Ile Leu Thr Arg180 185 190Val Leu Arg Thr Ser Ala Glu Lys Met Val Arg Leu Asn Tyr Glu Lys195 200 205Asn Tyr Val Gly Gly Thr Leu Ala Gly Ser Leu Arg Ser Ala Asn Ala210 215 220His Phe Ala Asn Met Leu Leu Gly Phe Tyr Leu Ala Thr Gly Gln Asp225 230 235 240Ala Ala Asn Ile Ile Glu Ala Ser Gln Gly Phe Val His Cys Glu Ala245 250 255Arg Gly Glu Asp Leu Tyr Phe Ser Cys Thr Leu Pro Asn Leu Ile Met260 265 270Gly Ser Val Gly Ala Gly Lys Gly Ile Pro Ser Ile Glu Glu Asn Leu275 280 285
Ser Arg Met Gly Cys Arg Gln Pro Gly Glu Pro Gly Asp Asn Ala Arg290 295 300Arg Leu Ala Ala Ile Cys Ala Gly Val Val Leu Cys Gly Glu Leu Ser305 310 315 320Leu Leu Ala Ala Gln Thr Asn Pro Gly Glu Leu Val Arg Thr His Met325 330 335Glu Met Glu Arg340<210>44<211>9066<212>DNA<213>副球菌属菌种(Paracoccus sp.)<220>
<221>CDS<222>(3641)..(4690)<223>
<400>44ggatccggca gctcgacacg ccgcagaacc tgtacgaacg tcccgccagc cgcttcgtcg 60cggaattcgt cgggcgcggg acggtggtgc ccgtgcaggc ccatgacggc gcgggccgcg 120cccgcatcct gggggccgag gtggcggtga acgccgcccc gcaatcgcgc tttgtcgatc 180acgtctgcct gcgccccgag aaccttgcca tctccgagac gggcgacctg cgcgccaagg 240tcgcgcgcgt cacctatctt ggcgggaaat acctgctgga aaccgtgctg gattgcggca 300cccggctggt gaccgagacc cgcgcccgct tcgatacggg cgcgcagctt ggcctgacca 360tcaacgcccc ctgggccttt gccgaggatt gaatggacag cgtgaagatc ctttcgggca 420tgggcgtgaa gggccctgcc tgcatcaggc tggatgtcgg cgggatgcgc ctgatcctcg 480attgcgggac cggcccggac gagggcgcgg agttcgaccc cgcctggctg gcggacgcgg 540atgcggtgct gatcacccat gaccacgtgg accatatcgg cggcgcgcgt cacgcggtcg 600cggcggggct gccgatccat gcgacgcggc agacggcggg gttgctgccc gcgggggcgg 660atctgcgcct gctgcccgaa cgcggtgtca cgcggatcgc cggggtcgat ctgacgaccg 720gtcgcaacgg gcatgccgcg ggcggcgtct ggatgcattt cgacatgggc gaggggctgt 780tctattccgg cgactggtcc gaggaatccg actggttcgc cttcgatccg cccccgcctg 840cggggacggc gattctcgac tgctcctatg gcggtttcga cgtggcgcaa tcggattgca 900tcgcggacct ggacgacctg ctcgaggtgc tgccggggca ggtactgctg ccggtgccgc 960catccggccg cgcggccgag ctggccctgc ggctgatccg ccgccacgga ccgggcagcg1020tgatggtcga cgacgcctgc ctgccggcca tcgcgcaact gcccgaggcg cgcggactgg1080cctacgccac cgaggcacgc tttcttgtct gcgacacgcc gaacgccgaa agccggcgcg1140gcatggcggc atctgcaagc atggcgcgat gcgggcaggc tggggcggga cgcgcatgtc1200gtcttcaccg ggcacatgaa cgtccatgcg cgcgcattct gcgaccgccc cggcgggcat1260ttccgccgct ggaacgtgca tccgccgctg cgcgaccagc gacggatgct ggaacggctg1320gccgcgcggc gctttgcccc ggccttctgc cccgaccccg agatctatct ggcgctggac1380atgggcgcgc aggtcttcat gcaccaggag gtgacgccat gatccccgcc cgcagcttct1440gcctgatccg ccacggcgaa acgaccgcca atgcaggggc gatcatcgcg ggcgcaaccg1500atgtgcccct gacgccaagg ggccgcgatc aggcccgcgc cctggcaggg cgcgaatggc1560catcgggcat cgcgctgttc gccagcccga tgtcgcgtgc ccgcgatacc gcgctgctgg1620cctttccggg gcgcgaccac cagcccgaac ccgatctgcg cgaacgcgac tggggcatct1680tcgagggacg ccccgtcgcc gatctgcccc cgcgcgaaat cacgccgcag gggggcgagg1740gctgggacga cgtgatggcc cgcgtggacc gcgcgatccg gcggatctgc gcgacctcgg1800gcgatgcgct gccggtgctg gtctgccatt cgggcgtgat ccgtgccgcg cgcgtgctgt1860ggaccaccgg cgatgcgggc gatcgtccgc ccaacgccac gccgatcctg ttcagcccgg1920acggcgaccg attaaaggaa ggaacgatat gaccgccacc accccctgcg tcgtcttcga1980acgtggacgg cacgcttgcc gaattcgacg ccgaccgcct gggccatctt gtccacggca2040cgaccaagca ctgggacgcc ttccaccacg cgatggccga cgccccgccc atccccgagg2100tcgcccgcct gatgcgcaag ctgaaggagg ggggcgagac ggtcgtcatc tgctcggggc2160ggccccgcgg ctggcaggat cagacgatcg catggctgcg caagcacgac ctgcccttcg2220acgggatcta tctgcgcccc gaggatcagg acggcgccag cgaccccgag gtcaagcgcc2280gcgccctagc cgagatgcgc gccgacgggc tggcgccctg gctggtcgtg gacgaccggc2340
ggtccgtcgt ggatgcctgg cgggccgagg ggctggtctg cctgcaatgc gcgccggggg2400acttctaggg ccgcgcgacg ggggcgcgga caggctgggc gggaaaccgc cccgccacca2460tgtcctgcac gcgtcgaacc gcccgtccga cgccggtttc cgcacggaaa cgcgcggcaa2520gttgacataa cttgcacgcg acgtctcgat tctgcccgcg aagaatgcga tgcatccaga2580tgatgcagaa cgaagaagcg gaagcgcccg tgaaagacca gatgatttcc cataccccgg2640tgcccacgca atgggtcggc ccgatcctgt tccgcggccc cgtcgtcgag ggcccgatca2700gcgcgccgct ggccacctac gagacgccgc tctggccctc gaccgcgcgg ggggcagggg2760tttcccggca ttcgggcggg atccaggtct cgctggtcga cgaacgcatg agccgctcga2820tcgcgctgcg ggcgcatgac ggggcggcgg cgaccgccgc ctggcagtcg atcaaggccc2880gccaggaaga ggtcgcggcc gtggtcgcca ccaccagccg cttcgcccgc cttgtcgagc2940tgaatcgcca gatcgtgggc aacctgcttt acatccgcat cgaatgcgtg acgggcgacg3000cctcgggtca caacatggtc accaaggccg ccgaggccgt gcagggctgg atcctgtcgg3060aatacccgat gctggcctat tccacgatct cggggaacct gtgcaccgac aagaaggcgt3120cggcggtcaa cggcatcctg ggccgcggca aatacgccgt cgccgaggtc gagatcccgc3180gcaagatcct gacccgcgtg ctgcgcacca gcgccgagaa gatggtccgc ctgaactacg3240agaagaacta tgtcgggggt acgctggcgg ggtcgctgcg cagtgcgaac gcgcatttcg3300ccaacatgct gctgggcttc tacctggcga cggggcagga cgcggccaac atcatcgagg3360ccagccaggg cttcgtccat tgcgaggccc gcggcgagga tctgtatttc tcgtgcacgc3420tgcccaacct catcatgggc tcggtcggtg ccggcaaggg catcccctcg atcgaggaga3480acctgtcgcg gatgggctgc cgccagccgg gcgaacccgg cgacaacgcg cgccgtcttg3540cggcgatctg cgcgggcgtc gtgctgtgtg gtgaattgtc gctgcttgcg gcccagacca3600accccggaga gttggtccgc acccacatgg agatggagcg atg acc gac agc aag 3655Met Thr Asp Ser Lys1 5gat cac cat gtc gcg ggg cgc aag ctg gac cat ctg cgt gca ttg gac 3703Asp His His Val Ala Gly Arg Lys Leu Asp His Leu Arg Ala Leu Asp10 15 20gac gat gcg gat atc gac cgg ggc gac agc ggc ttc gac cgc atc gcg 3751Asp Asp Ala Asp Ile Asp Arg Gly Asp Ser Gly Phe Asp Arg Ile Ala25 30 35ctg acc cat cgc gcc ctg ccc gag gtg gat ttc gac gcc atc gac acg 3799Leu Thr His Arg Ala Leu Pro Glu Val Asp Phe Asp Ala Ile Asp Thr40 45 50gcg acc agc ttc ctg ggc cgt gaa ctg tcc ttc ccg ctg ctg atc tcg 3847Ala Thr Ser Phe Leu Gly Arg Glu Leu Ser Phe Pro Leu Leu Ile Ser55 60 65tcc atg acc ggc ggc acc ggc gag gag atc gag cgc atc aac cgc aac 3895Ser Met Thr Gly Gly Thr Gly Glu Glu Ile Glu Arg Ile Asn Arg Asn70 75 80 85ctg gcc gct ggt gcc gag gag gcc cgc gtc gcc atg gcg gtg ggc tcg 3943Leu Ala Ala Gly Ala Glu Glu Ala Arg Val Ala Met Ala Val Gly Ser90 95 100cag cgc gtg atg ttc acc gac ccc tcg gcg cgg gcc agc ttc gac ctg 3991Gln Arg Val Met Phe Thr Asp Pro Ser Ala Arg Ala Ser Phe Asp Leu105 110 115cgc gcc cat gcg ccc acc gtg ccg ctg ctg gcc aat atc ggc gcg gtg 4039Arg Ala His Ala Pro Thr Val Pro Leu Leu Ala Asn Ile Gly Ala Val120 125 130cag ctg aac atg ggg ctg ggg ctg aag gaa tgc ctg gcc gcg atc gag 4087Gln Leu Asn Met Gly Leu Gly Leu Lys Glu Cys Leu Ala Ala Ile Glu
135 140 145gtg ctg cag gcg gac ggc ctg tat ctg cac ctg aac ccc ctg caa gag 4135Val Leu Gln Ala Asp Gly Leu Tyr Leu His Leu Asn pro Leu Gln Glu150 155 160 165gcc gtc cag ccc gag ggg gat cgc gac ttt gcc gat ctg ggc agc aag 4183Ala Val Gln Pro Glu Gly Asp Arg Asp Phe Ala Asp Leu Gly Ser Lys170 175 180atc gcg gcc atc gcc cgc gac gtt ccc gtg ccc gtc ctg ctg aag gag 4231Ile Ala Ala Ile Ala Arg Asp Val Pro Val Pro Val Leu Leu Lys Glu185 190 195gtg ggc tgc ggc ctg tcg gcg gcc gat atc gcc atc ggg ctg cgc gcc 4279Val Gly Cys Gly Leu Ser Ala Ala Asp Ile Ala Ile Gly Leu Arg Ala200 205 210ggg atc cgg cat ttc gac gtg gcc ggt cgc ggc ggc aca tcc tgg agc 4327Gly Ile Arg His Phe Asp Val Ala Gly Arg Gly Gly Thr Ser Trp Ser215 220 225cgg atc gag tat cgc cgc cgc cag cgg gcc gat gac gac ctg ggc ctg 4375Arg Ile Glu Tyr Arg Arg Arg Gln Arg Ala Asp Asp Asp Leu Gly Leu230 235 240 245gtc ttc cag gac tgg ggc ctg cag acc gtg gac gcc ctg cgc gag gcg 4423Val Phe Gln Asp Trp Gly Leu Gln Thr Val Asp Ala Leu Arg Glu Ala250 255 260cgg ccc gcg ctt gcg gcc cat gat gga acc agc gtg ctg atc gcc agc 4471Arg Pro Ala Leu Ala Ala His Asp Gly Thr Ser Val Leu Ile Ala Ser265 270 275ggc ggc atc cgc aac ggt gtc gac atg gcg aaa tgc gtc atc ctg ggg 4519Gly Gly Ile Arg Asn Gly Val Asp Met Ala Lys Cys Val Ile Leu Gly280 285 290gcc gac atg tgc ggg gtc gcc gcg ccc ctg ctg aaa gcg gcc caa aac 4567Ala Asp Met Cys Gly Val Ala Ala Pro Leu Leu Lys Ala Ala Gln Asn295 300 305tcg cgc gag gcg gtt gta tcc gcc atc cgg aaa ctg cat ctg gag ttc 4615Ser Arg Glu Ala Val Val Ser Ala Ile Arg Lys Leu His Leu Glu Phe310 315 320 325cgg aca gcc atg ttc ctc ctg ggt tgc ggc acg ctt gcc gac ctg aag 4663Arg Thr Ala Met Phe Leu Leu Gly Cys Gly Thr Leu Ala Asp Leu Lys330 335 340gac aat tcc tcg ctt atc cgt caa tga aagtgcctaa gatgaccgtg4710Asp Asn Ser Ser Leu Ile Arg Gln345acaggaatcg aagcgatcag cttctacacc ccccagaact acgtgggact ggatatcctt4770gccgcgcatc acgggatcga ccccgagaag ttctcgaagg ggatcgggca ggagaaaatc4830gcactgcccg gccatgacga ggatatcgtg accatggccg ccgaggccgc gctgccgatc4890atcgaacgcg cgggcacgca gggcatcgac acggttctgt tcgccaccga gagcgggatc4950gaccagtcga aggccgccgc catctatctg cgccgcctgc tggacctgtc gcccaactgc5010cgttgcgtcg agctgaagca ggcctgctat tccgcgacgg cggcgctgca gatggcctgc5070gcgcatgtcg cccgcaagcc cgaccgcaag gtgctggtga tcgcgtccga tgtcgcgcgc5130
tatgaccgcg aaagctcggg cgaggcgacg cagggtgcgg gcgccgtcgc catccttgtc5190agcgccgatc ccaaggtggc cgagatcggc accgtctcgg ggctgttcac cgaggatatc5250atggatttct ggcggccgaa ccaccgccgc acgcccctgt tcgacggcaa ggcatcgacg5310ctgcgctatc tgaacgcgct ggtcgaggcg tggaacgact atcgcgcgaa tggcggccac5370gagttcgccg atttcgcgca tttctgctat cacgtgccgt tctcgcggat gggcgagaag5430gcgaacagcc acctggccaa ggcgaacaag acgccggtgg acatggggca ggtgcagacg5490ggcctgatct acaaccggca ggtcgggaac tgctataccg ggtcgatcta cctggcattc5550gcctcgctgc tggagaacgc tcaggaggac ctgaccggcg cgctggtcgg tctgttcagc5610tatggctcgg gtgcgacggg cgaattcttc gatgcgcgga tcgcgcccgg ttaccgcgac5670cacctgttcg cggaacgcca tcgcgaattg ctgcaggatc gcacgcccgt cacatatgac5730gaatacgttg ccctgtggga cgagatcgac ctgacgcagg gcgcgcccga caaggcgcgc5790ggtcgtttca ggctggcagg tatcgaggac gagaagcgca tctatgtcga ccggcaggcc5850tgaagcaggc gcccatgccc cgggcaagct gatcctgtcc ggggaacatt ccgtgctcta5910tggtgcgccc gcgcttgcca tggccatcgc ccgctatacc gaggtgtggt tcacgccgct5970tggcattggc gaggggatac gcacgacatt cgccaatctc tcgggcgggg cgacctattc6030gctgaagctg ctgtcggggt tcaagtcgcg gctggaccgc cggttcgagc agttcctgaa6090cggcgaccta aaggtgcaca aggtcctgac ccatcccgac gatctggcgg tctatgcgct6150ggcgtcgctt ctgcacgaca agccgccggg gaccgccgcg atgccgggca tcggcgcgat6210gcaccacctg ccgcgaccgg gtgagctggg cagccggacg gagctgccca tcggcgcggg6270catggggtcg tctgcggcca tcgtcgcggc caccacggtc ctgttcgaga cgctgctgga6330ccggcccaag acgcccgaac agcgcttcga ccgcgtccgc ttctgcgagc ggttgaagca6390cggcaaggcc ggtcccatcg acgcggccag cgtcgtgcgc ggcgggcttg tccgcgtggg6450cgggaacggg ccgggttcga tcagcagctt cgatttgccc gaggatcacg accttgtcgc6510gggacgcggc tggtactggg tactgcacgg gcgccccgtc agcgggaccg gcgaatgcgt6570cagcgcggtc gcggcggcgc atggtcgcga tgcggcgctg tgggacgcct tcgcagtctg6630cacccgcgcg ttggaggccg cgctgctgtc tgggggcagc cccgacgccg ccatcaccga6690gaaccagcgc ctgctggaac gcatcggcgt cgtgccggca gcgacgcagg ccctcgtggc6750ccagatcgag gaggcgggtg gcgcggccaa gatctgcggc gcaggttccg tgcggggcga6810tcacggcggg gcggtcctcg tgcggattga cgacgcgcag gcgatggctt cggtcatggc6870gcgccatccc gacctcgact gggcgcccct gcgcatgtcg cgcacggggg cggcacccgg6930ccccgcgccg cgtgcgcaac cgctgccggg gcagggctga tggatcaggt catccgcgcc6990agcgcgccgg gttcggtcat gatcacgggc gaacatgccg tggtctatgg acaccgcgcc7050atcgtcgccg ggatcgagca gcgcgcccat gtgacgatcg tcccgcgtgc cgaccgcatg7110tttcgcatca cctcgcagat cggggcgccg cagcaggggt cgctggacga tctgcctgcg7170ggcgggacct atcgcttcgt gctggccgcc atcgcgcgac acgcgccgga cctgccttgc7230gggttcgaca tggacatcac ctcggggatc gatccgaggc tcgggcttgg atcctcggcg7290gcggtgacgg tcgcctgcct cggcgcgctg tcgcggctgg cggggcgggg gaccgagggg7350ctgcatgacg acgcgctgcg catcgtccgc gccatccagg gcaggggcag cggggccgat7410ctggcggcca gcctgcatgg cggcttcgtc gcctatcgcg cgcccgatgg cggtgccgcg7470cagatcgagg cgcttccggt gccgccgggg ccgttcggcc tgcgctatgc gggctacaag7530accccgacag ccgaggtgct gcgccttgtg gccgatcgga tggcgggcaa cgaggccgct7590ttcgacgcgc tctactcccg gatgggcgca agcgcagatg ccgcgatccg cgcggcgcaa7650gggctggact gggctgcatt ccacgacgcg ctgaacgaat accagcgcct gatggagcag7710ctgggcgtgt ccgacgacac gctggacgcg atcatccgcg aggcgcgcga cgcgggcgcc7770gcagtcgcca agatctccgg ctcggggctg ggggattgcg tgctggcact gggcgaccag7830cccaagggtt tcgtgcccgc aagcattgcc gagaagggac ttgttttcga tgactgatgc7890cgtccgcgac atgatcgccc gtgccatggc gggcgcgacc gacatccgag cagccgaggc7950ttatgcgccc agcaacatcg cgctgtcgaa atactggggc aagcgcgacg ccgcgcggaa8010ccttccgctg aacagctccg tctcgatctc gttggcgaac tggggctctc atacgcgggt8070cgaggggtcc ggcacgggcc acgacgaggt gcatcacaac ggcacgctgc tggatccggg8130cgacgccttc gcgcgccgcg cgttggcatt cgctgacctg ttccgggggg ggaggcacct8190gccgctgcgg atcacgacgc agaactcgat cccgacggcg gcggggcttg cctcgtcggc8250ctcggggttc gcggcgctga cccgtgcgct ggcgggggcg ttcgggctgg atctggacga8310cacggatctg agccgcatcg cccggatcgg cagtggcagc gccgcccgct cgatctggca8370cggcttcgtc cgctggaacc ggggcgaggc cgaggatggg catgacagcc acggcgtccc8430gctggacctg cgctggcccg gcttccgcat cgcgatcgtg gccgtggaca aggggcccaa8490gcctttcagt tcgcgcgacg gcatgaacca cacggtcgag accagcccgc tgttcccgcc8550ctggcctgcg caggcggaag cggattgccg cgtcatcgag gatgcgatcg ccgcccgcga8610catggccgcc ctgggtccgc gggtcgaggc gaacgccctt gcgatgcacg ccacgatgat8670ggccgcgcgc ccgccgctct gctacctgac gggcggcagc tggcaggtgc tggaacgcct8730
gtggcaggcc cgcgcggacg ggcttgcggc ctttgcgacg atggatgccg gcccgaacgt8790caagctgatc ttcgaggaaa gcagcgccgc cgacgtgctg tacctgttcc ccgacgccag8850cctgatcgcg ccgttcgagg ggcgttgaac gcgtaagacg accactgggt aaggttctgc8910cgcgcgtggt ctcgactgcc tgcaaagagg tgcttgagtt gctgcgtgac tgcggcggcc8970gacttcgtgg gacttgcccg ccacgctgac gcgctggaaa cgcgcccgcg gattacgacc9030gcgtcattgc cctgaaccaa tttcccgtcg gtcgac 9066<210>45<211>349<212>PRT<213>副球菌属菌种(Paracoccus sp.)<400>45Met Thr Asp Ser Lys Asp His His Val Ala Gly Arg Lys Leu Asp His1 5 10 15Leu Arg Ala Leu Asp Asp Asp Ala Asp Ile Asp Arg Gly Asp Ser Gly20 25 30Phe Asp Arg Ile Ala Leu Thr His Arg Ala Leu Pro Glu Val Asp Phe35 40 45Asp Ala Ile Asp Thr Ala Thr Ser Phe Leu Gly Arg Glu Leu Ser Phe50 55 60Pro Leu Leu Ile Ser Ser Met Thr Gly Gly Thr Gly Glu Glu Ile Glu65 70 75 80Arg Ile Asn Arg Asn Leu Ala Ala Gly Ala Glu Glu Ala Arg Val Ala85 90 95Met Ala Val Gly Ser Gln Arg Val Met Phe Thr Asp Pro Ser Ala Arg100 105 110Ala Ser Phe Asp Leu Arg Ala His Ala Pro Thr Val Pro Leu Leu Ala115 120 125Asn Ile Gly Ala Val Gln Leu Asn Met Gly Leu Gly Leu Lys Glu Cys130 135 140Leu Ala Ala Ile Glu Val Leu Gln Ala Asp Gly Leu Tyr Leu His Leu145 150 155 160Asn Pro Leu Gln Glu Ala Val Gln Pro Glu Gly Asp Arg Asp Phe Ala165 170 175Asp Leu Gly Ser Lys Ile Ala Ala Ile Ala Arg Asp Val Pro Val Pro180 185 190Val Leu Leu Lys Glu Val Gly Cys Gly Leu Ser Ala Ala Asp Ile Ala195 200 205Ile Gly Leu Arg Ala Gly Ile Arg His Phe Asp Val Ala Gly Arg Gly210 215 220Gly Thr Ser Trp Ser Arg Ile Glu Tyr Arg Arg Arg Gln Arg Ala Asp225 230 235 240Asp Asp Leu Gly Leu Val Phe Gln Asp Trp Gly Leu Gln Thr Val Asp245 250 255
Ala Leu Arg Glu Ala Arg Pro Ala Leu Ala Ala His Asp Gly Thr Ser260 265 270Val Leu Ile Ala Ser Gly Gly Ile Arg Asn Gly Val Asp Met Ala Lys275 280 285Cys Val Ile Leu Gly Ala Asp Met Cys Gly Val Ala Ala Pro Leu Leu290 295 300Lys Ala Ala Gln Asn Ser Arg Glu Ala Val Val Ser Ala Ile Arg Lys305 310 315 320Leu His Leu Glu Phe Arg Thr Ala Met Phe Leu Leu Gly Cys Gly Thr325 330 335Leu Ala Asp Leu Lys Asp Asn Ser Ser Leu Ile Arg Gln340 345<210>46<211>9066<212>DNA<213>副球菌属菌种(Paracoccus sp.)<220>
<221>CDS<222>(4687)..(5853)<223>
<400>46ggatccggca gctcgacacg ccgcagaacc tgtacgaacg tcccgccagc cgcttcgtcg 60cggaattcgt cgggcgcggg acggtggtgc ccgtgcaggc ccatgacggc gcgggccgcg 120cccgcatcct gggggccgag gtggcggtga acgccgcccc gcaatcgcgc tttgtcgatc 180acgtctgcct gcgccccgag aaccttgcca tctccgagac gggcgacctg cgcgccaagg 240tcgcgcgcgt cacctatctt ggcgggaaat acctgctgga aaccgtgctg gattgcggca 300cccggctggt gaccgagacc cgcgcccgct tcgatacggg cgcgcagctt ggcctgacca 360tcaacgcccc ctgggccttt gccgaggatt gaatggacag cgtgaagatc ctttcgggca 420tgggcgtgaa gggccctgcc tgcatcaggc tggatgtcgg cgggatgcgc ctgatcctcg 480attgcgggac cggcccggac gagggcgcgg agttcgaccc cgcctggctg gcggacgcgg 540atgcggtgct gatcacccat gaccacgtgg accatatcgg cggcgcgcgt cacgcggtcg 600cggcggggct gccgatccat gcgacgcggc agacggcggg gttgctgccc gcgggggcgg 660atctgcgcct gctgcccgaa cgcggtgtca cgcggatcgc cggggtcgat ctgacgaccg 720gtcgcaacgg gcatgccgcg ggcggcgtct ggatgcattt cgacatgggc gaggggctgt 780tctattccgg cgactggtcc gaggaatccg actggttcgc cttcgatccg cccccgcctg 840cggggacggc gattctcgac tgctcctatg gcggtttcga cgtggcgcaa tcggattgca 900tcgcggacct ggacgacctg ctcgaggtgc tgccggggca ggtactgctg ccggtgccgc 960catccggccg cgcggccgag ctggccctgc ggctgatccg ccgccacgga ccgggcagcg1020tgatggtcga cgacgcctgc ctgccggcca tcgcgcaact gcccgaggcg cgcggactgg1080cctacgccac cgaggcacgc tttcttgtct gcgacacgcc gaacgccgaa agccggcgcg1140gcatggcggc atctgcaagc atggcgcgat gcgggcaggc tggggcggga cgcgcatgtc1200gtcttcaccg ggcacatgaa cgtccatgcg cgcgcattct gcgaccgccc cggcgggcat1260ttccgccgct ggaacgtgca tccgccgctg cgcgaccagc gacggatgct ggaacggctg1320gccgcgcggc gctttgcccc ggccttctgc cccgaccccg agatctatct ggcgctggac1380atgggcgcgc aggtcttcat gcaccaggag gtgacgccat gatccccgcc cgcagcttct1440gcctgatccg ccacggcgaa acgaccgcca atgcaggggc gatcatcgcg ggcgcaaccg1500atgtgcccct gacgccaagg ggccgcgatc aggcccgcgc cctggcaggg cgcgaatggc1560catcgggcat cgcgctgttc gccagcccga tgtcgcgtgc ccgcgatacc gcgctgctgg1620cctttccggg gcgcgaccac cagcccgaac ccgatctgcg cgaacgcgac tggggcatct1680tcgagggacg ccccgtcgcc gatctgcccc cgcgcgaaat cacgccgcag gggggcgagg1740gctgggacga cgtgatggcc cgcgtggacc gcgcgatccg gcggatctgc gcgacctcgg1800gcgatgcgct gccggtgctg gtctgccatt cgggcgtgat ccgtgccgcg cgcgtgctgt1860
ggaccaccgg cgatgcgggc gatcgtccgc ccaacgccac gccgatcctg ttcagcccgg1920acggcgaccg attaaaggaa ggaacgatat gaccgccacc accccctgcg tcgtcttcga1980acgtggacgg cacgcttgcc gaattcgacg ccgaccgcct gggccatctt gtccacggca2040cgaccaagca ctgggacgcc ttccaccacg cgatggccga cgccccgccc atccccgagg2100tcgcccgcct gatgcgcaag ctgaaggagg ggggcgagac ggtcgtcatc tgctcggggc2160ggccccgcgg ctggcaggat cagacgatcg catggctgcg caagcacgac ctgcccttcg2220acgggatcta tctgcgcccc gaggatcagg acggcgccag cgaccccgag gtcaagcgcc2280gcgccctagc cgagatgcgc gccgacgggc tggcgccctg gctggtcgtg gacgaccggc2340ggtccgtcgt ggatgcctgg cgggccgagg ggctggtctg cctgcaatgc gcgccggggg2400acttctaggg ccgcgcgacg ggggcgcgga caggctgggc gggaaaccgc cccgccacca2460tgtcctgcac gcgtcgaacc gcccgtccga cgccggtttc cgcacggaaa cgcgcggcaa2520gttgacataa cttgcacgcg acgtctcgat tctgcccgcg aagaatgcga tgcatccaga2580tgatgcagaa cgaagaagcg gaagcgcccg tgaaagacca gatgatttcc cataccccgg2640tgcccacgca atgggtcggc ccgatcctgt tccgcggccc cgtcgtcgag ggcccgatca2700gcgcgccgct ggccacctac gagacgccgc tctggccctc gaccgcgcgg ggggcagggg2760tttcccggca ttcgggcggg atccaggtct cgctggtcga cgaacgcatg agccgctcga2820tcgcgctgcg ggcgcatgac ggggcggcgg cgaccgccgc ctggcagtcg atcaaggccc2880gccaggaaga ggtcgcggcc gtggtcgcca ccaccagccg cttcgcccgc cttgtcgagc2940tgaatcgcca gatcgtgggc aacctgcttt acatccgcat cgaatgcgtg acgggcgacg3000cctcgggtca caacatggtc accaaggccg ccgaggccgt gcagggctgg atcctgtcgg3060aatacccgat gctggcctat tccacgatct cggggaacct gtgcaccgac aagaaggcgt3120cggcggtcaa cggcatcctg ggccgcggca aatacgccgt cgccgaggtc gagatcccgc3180gcaagatcct gacccgcgtg ctgcgcacca gcgccgagaa gatggtccgc ctgaactacg3240agaagaacta tgtcgggggt acgctggcgg ggtcgctgcg cagtgcgaac gcgcatttcg3300ccaacatgct gctgggcttc tacctggcga cggggcagga cgcggccaac atcatcgagg3360ccagccaggg cttcgtccat tgcgaggccc gcggcgagga tctgtatttc tcgtgcacgc3420tgcccaacct catcatgggc tcggtcggtg ccggcaaggg catcccctcg atcgaggaga3480acctgtcgcg gatgggctgc cgccagccgg gcgaacccgg cgacaacgcg cgccgtcttg3540cggcgatctg cgcgggcgtc gtgctgtgtg gtgaattgtc gctgcttgcg gcccagacca3600accccggaga gttggtccgc acccacatgg agatggagcg atgaccgaca gcaaggatca3660ccatgtcgcg gggcgcaagc tggaccatct gcgtgcattg gacgacgatg cggatatcga3720ccggggcgac agcggcttcg accgcatcgc gctgacccat cgcgccctgc ccgaggtgga3780tttcgacgcc atcgacacgg cgaccagctt cctgggccgt gaactgtcct tcccgctgct3840gatctcgtcc atgaccggcg gcaccggcga ggagatcgag cgcatcaacc gcaacctggc3900cgctggtgcc gaggaggccc gcgtcgccat ggcggtgggc tcgcagcgcg tgatgttcac3960cgacccctcg gcgcgggcca gcttcgacct gcgcgcccat gcgcccaccg tgccgctgct4020ggccaatatc ggcgcggtgc agctgaacat ggggctgggg ctgaaggaat gcctggccgc4080gatcgaggtg ctgcaggcgg acggcctgta tctgcacctg aaccccctgc aagaggccgt4140ccagcccgag ggggatcgcg actttgccga tctgggcagc aagatcgcgg ccatcgcccg4200cgacgttccc gtgcccgtcc tgctgaagga ggtgggctgc ggcctgtcgg cggccgatat4260cgccatcggg ctgcgcgccg ggatccggca tttcgacgtg gccggtcgcg gcggcacatc4320ctggagccgg atcgagtatc gccgccgcca gcgggccgat gacgacctgg gcctggtctt4380ccaggactgg ggcctgcaga ccgtggacgc cctgcgcgag gcgcggcccg cgcttgcggc4440ccatgatgga accagcgtgc tgatcgccag cggcggcatc cgcaacggtg tcgacatggc4500gaaatgcgtc atcctggggg ccgacatgtg cggggtcgcc gcgcccctgc tgaaagcggc4560ccaaaactcg cgcgaggcgg ttgtatccgc catccggaaa ctgcatctgg agttccggac4620agccatgttc ctcctgggtt gcggcacgct tgccgacctg aaggacaatt cctcgcttat4680ccgtca atg aaa gtg cct aag atg acc gtg aca gga atc gaa gcg atc 4728Met Lys Val Pro Lys Met Thr Val Thr Gly Ile Glu Ala Ile1 5 10agc ttc tac acc ccc cag aac tac gtg gga ctg gat atc ctt gcc gcg 4776Ser Phe Tyr Thr Pro Gln Asn Tyr Val Gly Leu Asp Ile Leu Ala Ala15 20 25 30cat cac ggg atc gac ccc gag aag ttc tcg aag ggg atc ggg cag gag 4824His His Gly Ile Asp Pro Glu Lys Phe Ser Lys Gly Ile Gly Gln Glu35 40 45aaa atc gca ctg ccc ggc cat gac gag gat atc gtg acc atg gcc gcc 4872
Lys Ile Ala Leu Pro Gly His Asp Glu Asp Ile Val Thr Met Ala Ala50 55 60gag gcc gcg ctg ccg atc atc gaa cgc gcg ggc acg cag ggc atc gac 4920Glu Ala Ala Leu Pro Ile Ile Glu Arg Ala Gly Thr Gln Gly Ile Asp65 70 75acg gtt ctg ttc gcc acc gag agc ggg atc gac cag tcg aag gcc gcc 4968Thr Val Leu Phe Ala Thr Glu Ser Gly Ile Asp Gln Ser Lys Ala Ala80 85 90gcc atc tat ctg cgc cgc ctg ctg gac ctg tcg ccc aac tgc cgt tgc 5016Ala Ile Tyr Leu Arg Arg Leu Leu Asp Leu Ser Pro Asn Cys Arg Cys95 100 105 110gtc gag ctg aag cag gcc tgc tat tcc gcg acg gcg gcg ctg cag atg 5064Val Glu Leu Lys Gln Ala Cys Tyr Ser Ala Thr Ala Ala Leu Gln Met115 120 125gcc tgc gcg cat gtc gcc cgc aag ccc gac cgc aag gtg ctg gtg atc 5112Ala Cys Ala His Val Ala Arg Lys Pro Asp Arg Lys Val Leu Val Ile130 135 140gcg tcc gat gtc gcg cgc tat gac cgc gaa agc tcg ggc gag gcg acg 5160Ala Ser Asp Val Ala Arg Tyr Asp Arg Glu Ser Ser Gly Glu Ala Thr145 150 155cag ggt gcg ggc gcc gtc gcc atc ctt gtc agc gcc gat ccc aag gtg 5208Gln Gly Ala Gly Ala Val Ala Ile Leu Val Ser Ala Asp Pro Lys Val160 165 170gcc gag atc ggc acc gtc tcg ggg ctg ttc acc gag gat atc atg gat 5256Ala Glu Ile Gly Thr Val Ser Gly Leu Phe Thr Glu Asp Ile Met Asp175 180 185 190ttc tgg cgg ccg aac cac cgc cgc acg ccc ctg ttc gac ggc aag gca 5304Phe Trp Arg Pro Asn His Arg Arg Thr Pro Leu Phe Asp Gly Lys Ala195 200 205tcg acg ctg cgc tat ctg aac gcg ctg gtc gag gcg tgg aac gac tat 5352Ser Thr Leu Arg Tyr Leu Asn Ala Leu Val Glu Ala Trp Asn Asp Tyr210 215 220cgc gcg aat ggc ggc cac gag ttc gcc gat ttc gcg cat ttc tgc tat 5400Arg Ala Asn Gly Gly His Glu Phe Ala Asp Phe Ala His Phe Cys Tyr225 230 235cac gtg ccg ttc tcg cgg atg ggc gag aag gcg aac agc cac ctg gcc 5448His Val Pro Phe Ser Arg Met Gly Glu Lys Ala Asn Ser His Leu Ala240 245 250aag gcg aac aag acg ccg gtg gac atg ggg cag gtg cag acg ggc ctg 5496Lys Ala Asn Lys Thr Pro Val Asp Met Gly Gln Val Gln Thr Gly Leu255 260 265 270atc tac aac cgg cag gtc ggg aac tgc tat acc ggg tcg atc tac ctg 5544Ile Tyr Asn Arg Gln Val Gly Asn Cys Tyr Thr Gly Ser Ile Tyr Leu275 280 285gca ttc gcc tcg ctg ctg gag aac gct cag gag gac ctg acc ggc gcg 5592Ala Phe Ala Ser Leu Leu Glu Asn Ala Gln Glu Asp Leu Thr Gly Ala
290 295 300ctg gtc ggt ctg ttc agc tat ggc tcg ggt gcg acg ggc gaa ttc ttc 5640Leu Val Gly Leu Phe Ser Tyr Gly Ser Gly Ala Thr Gly Glu Phe Phe305 310 315gat gcg cgg atc gcg ccc ggt tac cgc gac cac ctg ttc gcg gaa cgc 5688Asp Ala Arg Ile Ala Pro Gly Tyr Arg Asp His Leu Phe Ala Glu Arg320 325 330cat cgc gaa ttg ctg cag gat cgc acg ccc gtc aca tat gac gaa tac 5736His Arg Glu Leu Leu Gln Asp Arg Thr Pro Val Thr Tyr Asp Glu Tyr335 340 345 350gtt gcc ctg tgg gac gag atc gac ctg acg cag ggc gcg ccc gac aag 5784Val Ala Leu Trp Asp Glu Ile Asp Leu Thr Gln Gly Ala Pro Asp Lys355 360 365gcg cgc ggt cgt ttc agg ctg gca ggt atc gag gac gag aag cgc atc 5832Ala Arg Gly Arg Phe Arg Leu Ala Gly Ile Glu Asp Glu Lys Arg Ile370 375 380tat gtc gac cgg cag gcc tga agcaggcgcc catgccccgg gcaagctgat 5883Tyr Val Asp Arg Gln Ala385cctgtccggg gaacattccg tgctctatgg tgcgcccgcg cttgccatgg ccatcgcccg5943ctataccgag gtgtggttca cgccgcttgg cattggcgag gggatacgca cgacattcgc6003caatctctcg ggcggggcga cctattcgct gaagctgctg tcggggttca agtcgcggct6063ggaccgccgg ttcgagcagt tcctgaacgg cgacctaaag gtgcacaagg tcctgaccca6123tcccgacgat ctggcggtct atgcgctggc gtcgcttctg cacgacaagc cgccggggac6183cgccgcgatg ccgggcatcg gcgcgatgca ccacctgccg cgaccgggtg agctgggcag6243ccggacggag ctgcccatcg gcgcgggcat ggggtcgtct gcggccatcg tcgcggccac6303cacggtcctg ttcgagacgc tgctggaccg gcccaagacg cccgaacagc gcttcgaccg6363cgtccgcttc tgcgagcggt tgaagcacgg caaggccggt cccatcgacg cggccagcgt6423cgtgcgcggc gggcttgtcc gcgtgggcgg gaacgggccg ggttcgatca gcagcttcga6483tttgcccgag gatcacgacc ttgtcgcggg acgcggctgg tactgggtac tgcacgggcg6543ccccgtcagc gggaccggcg aatgcgtcag cgcggtcgcg gcggcgcatg gtcgcgatgc6603ggcgctgtgg gacgccttcg cagtctgcac ccgcgcgttg gaggccgcgc tgctgtctgg6663gggcagcccc gacgccgcca tcaccgagaa ccagcgcctg ctggaacgca tcggcgtcgt6723gccggcagcg acgcaggccc tcgtggccca gatcgaggag gcgggtggcg cggccaagat6783ctgcggcgca ggttccgtgc ggggcgatca cggcggggcg gtcctcgtgc ggattgacga6843cgcgcaggcg atggcttcgg tcatggcgcg ccatcccgac ctcgactggg cgcccctgcg6903catgtcgcgc acgggggcgg cacccggccc cgcgccgcgt gcgcaaccgc tgccggggca6963gggctgatgg atcaggtcat ccgcgccagc gcgccgggtt cggtcatgat cacgggcgaa7023catgccgtgg tctatggaca ccgcgccatc gtcgccggga tcgagcagcg cgcccatgtg7083acgatcgtcc cgcgtgccga ccgcatgttt cgcatcacct cgcagatcgg ggcgccgcag7143caggggtcgc tggacgatct gcctgcgggc gggacctatc gcttcgtgct ggccgccatc7203gcgcgacacg cgccggacct gccttgcggg ttcgacatgg acatcacctc ggggatcgat7263ccgaggctcg ggcttggatc ctcggcggcg gtgacggtcg cctgcctcgg cgcgctgtcg7323cggctggcgg ggcgggggac cgaggggctg catgacgacg cgctgcgcat cgtccgcgcc7383atccagggca ggggcagcgg ggccgatctg gcggccagcc tgcatggcgg cttcgtcgcc7443tatcgcgcgc ccgatggcgg tgccgcgcag atcgaggcgc ttccggtgcc gccggggccg7503ttcggcctgc gctatgcggg ctacaagacc ccgacagccg aggtgctgcg ccttgtggcc7563gatcggatgg cgggcaacga ggccgctttc gacgcgctct actcccggat gggcgcaagc7623gcagatgccg cgatccgcgc ggcgcaaggg ctggactggg ctgcattcca cgacgcgctg7683aacgaatacc agcgcctgat ggagcagctg ggcgtgtccg acgacacgct ggacgcgatc7743atccgcgagg cgcgcgacgc gggcgccgca gtcgccaaga tctccggctc ggggctgggg7803gattgcgtgc tggcactggg cgaccagccc aagggtttcg tgcccgcaag cattgccgag7863aagggacttg ttttcgatga ctgatgccgt ccgcgacatg atcgcccgtg ccatggcggg7923cgcgaccgac atccgagcag ccgaggctta tgcgcccagc aacatcgcgc tgtcgaaata7983
ctggggcaag cgcgacgccg cgcggaacct tccgctgaac agctccgtct cgatctcgtt8043ggcgaactgg ggctctcata cgcgggtcga ggggtccggc acgggccacg acgaggtgca8103tcacaacggc acgctgctgg atccgggcga cgccttcgcg cgccgcgcgt tggcattcgc8163tgacctgttc cgggggggga ggcacctgcc gctgcggatc acgacgcaga actcgatccc8223gacggcggcg gggcttgcct cgtcggcctc ggggttcgcg gcgctgaccc gtgcgctggc8283gggggcgttc gggctggatc tggacgacac ggatctgagc cgcatcgccc ggatcggcag8343tggcagcgcc gcccgctcga tctggcacgg cttcgtccgc tggaaccggg gcgaggccga8403ggatgggcat gacagccacg gcgtcccgct ggacctgcgc tggcccggct tccgcatcgc8463gatcgtggcc gtggacaagg ggcccaagcc tttcagttcg cgcgacggca tgaaccacac8523ggtcgagacc agcccgctgt tcccgccctg gcctgcgcag gcggaagcgg attgccgcgt8583catcgaggat gcgatcgccg cccgcgacat ggccgccctg ggtccgcggg tcgaggcgaa8643cgcccttgcg atgcacgcca cgatgatggc cgcgcgcccg ccgctctgct acctgacggg8703cggcagctgg caggtgctgg aacgcctgtg gcaggcccgc gcggacgggc ttgcggcctt8763tgcgacgatg gatgccggcc cgaacgtcaa gctgatcttc gaggaaagca gcgccgccga8823cgtgctgtac ctgttccccg acgccagcct gatcgcgccg ttcgaggggc gttgaacgcg8883taagacgacc actgggtaag gttctgccgc gcgtggtctc gactgcctgc aaagaggtgc8943ttgagttgct gcgtgactgc ggcggccgac ttcgtgggac ttgcccgcca cgctgacgcg9003ctggaaacgc gcccgcggat tacgaccgcg tcattgccct gaaccaattt cccgtcggtc9063gac 9066<210>47<211>388<212>PRT<213>副球菌属菌种(Paracoccus sp.)<400>47Met Lys Val Pro Lys Met Thr Val Thr Gly Ile Glu Ala Ile Ser Phe1 5 10 15Tyr Thr Pro Gln Asn Tyr Val Gly Leu Asp Ile Leu Ala Ala His His20 25 30Gly Ile Asp Pro Glu Lys Phe Ser Lys Gly Ile Gly Gln Glu Lys Ile35 40 45Ala Leu Pro Gly His Asp Glu Asp Ile Val Thr Met Ala Ala Glu Ala50 55 60Ala Leu Pro Ile Ile Glu Arg Ala Gly Thr Gln Gly Ile Asp Thr Val65 70 75 80Leu Phe Ala Thr Glu Ser Gly Ile Asp Gln Ser Lys Ala Ala Ala Ile85 90 95Tyr Leu Arg Arg Leu Leu Asp Leu Ser Pro Asn Cys Arg Cys Val Glu100 105 110Leu Lys Gln Ala Cys Tyr Ser Ala Thr Ala Ala Leu Gln Met Ala Cys115 120 125Ala His Val Ala Arg Lys Pro Asp Arg Lys Val Leu Val Ile Ala Ser130 135 140Asp Val Ala Arg Tyr Asp Arg Glu Ser Ser Gly Glu Ala Thr Gln Gly145 150 155 160Ala Gly Ala Val Ala Ile Leu Val Ser Ala Asp Pro Lys Val Ala Glu165 170 175Ile Gly Thr Val Ser Gly Leu Phe Thr Glu Asp Ile Met Asp Phe Trp
180 185 190Arg Pro Asn His Arg Arg Thr Pro Leu Phe Asp Gly Lys Ala Ser Thr195 200 205Leu Arg Tyr Leu Asn Ala Leu Val Glu Ala Trp Asn Asp Tyr Arg Ala210 215 220Asn Gly Gly His Glu Phe Ala Asp Phe Ala His Phe Cys Tyr His Val225 230 235 240Pro Phe Ser Arg Met Gly Glu Lys Ala Asn Ser His Leu Ala Lys Ala245 250 255Asn Lys Thr Pro Val Asp Met Gly Gln Val Gln Thr Gly Leu Ile Tyr260 265 270Asn Arg Gln Val Gly Asn Cys Tyr Thr Gly Ser Ile Tyr Leu Ala Phe275 280 285ALa Ser Leu Leu Glu Asn Ala Gln Glu Asp Leu Thr Gly Ala Leu Val290 295 300Gly Leu Phe Ser Tyr Gly Ser Gly Ala Thr Gly Glu Phe Phe Asp Ala305 310 315 320Arg Ile Ala Pro Gly Tyr Arg Asp His Leu Phe Ala Glu Arg His Arg325 330 335Glu Leu Leu Gln Asp Arg Thr Pro Val Thr Tyr Asp Glu Tyr Val Ala340 345 350Leu Trp Asp Glu Ile Asp Leu Thr Gln Gly Ala Pro Asp Lys Ala Arg355 360 365Gly Arg Phe Arg Leu Ala Gly Ile Glu Asp Glu Lys Arg Ile Tyr Val370 375 380Asp Arg Gln Ala385<210>48<211>9066<212>DNA<213>副球菌属菌种(Paracoccus sp.)<220>
<221>CDS<222>(5834)..(6970)<223>
<400>48ggatccggca gctcgacacg ccgcagaacc tgtacgaacg tcccgccagc cgcttcgtcg 60cggaattcgt cgggcgcggg acggtggtgc ccgtgcaggc ccatgacggc gcgggccgcg120cccgcatcct gggggccgag gtggcggtga acgccgcccc gcaatcgcgc tttgtcgatc180acgtctgcct gcgccccgag aaccttgcca tctccgagac gggcgacctg cgcgccaagg240tcgcgcgcgt cacctatctt ggcgggaaat acctgctgga aaccgtgctg gattgcggca300cccggctggt gaccgagacc cgcgcccgct tcgatacggg cgcgcagctt ggcctgacca360tcaacgcccc ctgggccttt gccgaggatt gaatggacag cgtgaagatc ctttcgggca420tgggcgtgaa gggccctgcc tgcatcaggc tggatgtcgg cgggatgcgc ctgatcctcg480attgcgggac cggcccggac gagggcgcgg agttcgaccc cgcctggctg gcggacgcgg540
atgcggtgct gatcacccat gaccacgtgg accatatcgg cggcgcgcgt cacgcggtcg 600cggcggggct gccgatccat gcgacgcggc agacggcggg gttgctgccc gcgggggcgg 660atctgcgcct gctgcccgaa cgcggtgtca cgcggatcgc cggggtcgat ctgacgaccg 720gtcgcaacgg gcatgccgcg ggcggcgtct ggatgcattt cgacatgggc gaggggctgt 780tctattccgg cgactggtcc gaggaatccg actggttcgc cttcgatccg cccccgcctg 840cggggacggc gattctcgac tgctcctatg gcggtttcga cgtggcgcaa tcggattgca 900tcgcggacct ggacgacctg ctcgaggtgc tgccggggca ggtactgctg ccggtgccgc 960catccggccg cgcggccgag ctggccctgc ggctgatccg ccgccacgga ccgggcagcg1020tgatggtcga cgacgcctgc ctgccggcca tcgcgcaact gcccgaggcg cgcggactgg1080cctacgccac cgaggcacgc tttcttgtct gcgacacgcc gaacgccgaa agccggcgcg1140gcatggcggc atctgcaagc atggcgcgat gcgggcaggc tggggcggga cgcgcatgtc1200gtcttcaccg ggcacatgaa cgtccatgcg cgcgcattct gcgaccgccc cggcgggcat1260ttccgccgct ggaacgtgca tccgccgctg cgcgaccagc gacggatgct ggaacggctg1320gccgcgcggc gctttgcccc ggccttctgc cccgaccccg agatctatct ggcgctggac1380atgggcgcgc aggtcttcat gcaccaggag gtgacgccat gatccccgcc cgcagcttct1440gcctgatccg ccacggcgaa acgaccgcca atgcaggggc gatcatcgcg ggcgcaaccg1500atgtgcccct gacgccaagg ggccgcgatc aggcccgcgc cctggcaggg cgcgaatggc1560catcgggcat cgcgctgttc gccagcccga tgtcgcgtgc ccgcgatacc gcgctgctgg1620cctttccggg gcgcgaccac cagcccgaac ccgatctgcg cgaacgcgac tggggcatct1680tcgagggacg ccccgtcgcc gatctgcccc cgcgcgaaat cacgccgcag gggggcgagg1740gctgggacga cgtgatggcc cgcgtggacc gcgcgatccg gcggatctgc gcgacctcgg1800gcgatgcgct gccggtgctg gtctgccatt cgggcgtgat ccgtgccgcg cgcgtgctgt1860ggaccaccgg cgatgcgggc gatcgtccgc ccaacgccac gccgatcctg ttcagcccgg1920acggcgaccg attaaaggaa ggaacgatat gaccgccacc accccctgcg tcgtcttcga1980acgtggacgg cacgcttgcc gaattcgacg ccgaccgcct gggccatctt gtccacggca2040cgaccaagca ctgggacgcc ttccaccacg cgatggccga cgccccgccc atccccgagg2100tcgcccgcct gatgcgcaag ctgaaggagg ggggcgagac ggtcgtcatc tgctcggggc2160ggccccgcgg ctggcaggat cagacgatcg catggctgcg caagcacgac ctgcccttcg2220acgggatcta tctgcgcccc gaggatcagg acggcgccag cgaccccgag gtcaagcgcc2280gcgccctagc cgagatgcgc gccgacgggc tggcgccctg gctggtcgtg gacgaccggc2340ggtccgtcgt ggatgcctgg cgggccgagg ggctggtctg cctgcaatgc gcgccggggg2400acttctaggg ccgcgcgacg ggggcgcgga caggctgggc gggaaaccgc cccgccacca2460tgtcctgcac gcgtcgaacc gcccgtccga cgccggtttc cgcacggaaa cgcgcggcaa2520gttgacataa cttgcacgcg acgtctcgat tctgcccgcg aagaatgcga tgcatccaga2580tgatgcagaa cgaagaagcg gaagcgcccg tgaaagacca gatgatttcc cataccccgg2640tgcccacgca atgggtcggc ccgatcctgt tccgcggccc cgtcgtcgag ggcccgatca2700gcgcgccgct ggccacctac gagacgccgc tctggccctc gaccgcgcgg ggggcagggg2760tttcccggca ttcgggcggg atccaggtct cgctggtcga cgaacgcatg agccgctcga2820tcgcgctgcg ggcgcatgac ggggcggcgg cgaccgccgc ctggcagtcg atcaaggccc2880gccaggaaga ggtcgcggcc gtggtcgcca ccaccagccg cttcgcccgc cttgtcgagc2940tgaatcgcca gatcgtgggc aacctgcttt acatccgcat cgaatgcgtg acgggcgacg3000cctcgggtca caacatggtc accaaggccg ccgaggccgt gcagggctgg atcctgtcgg3060aatacccgat gctggcctat tccacgatct cggggaacct gtgcaccgac aagaaggcgt3120cggcggtcaa cggcatcctg ggccgcggca aatacgccgt cgccgaggtc gagatcccgc3180gcaagatcct gacccgcgtg ctgcgcacca gcgccgagaa gatggtccgc ctgaactacg3240agaagaacta tgtcgggggt acgctggcgg ggtcgctgcg cagtgcgaac gcgcatttcg3300ccaacatgct gctgggcttc tacctggcga cggggcagga cgcggccaac atcatcgagg3360ccagccaggg cttcgtccat tgcgaggccc gcggcgagga tctgtatttc tcgtgcacgc3420tgcccaacct catcatgggc tcggtcggtg ccggcaaggg catcccctcg atcgaggaga3480acctgtcgcg gatgggctgc cgccagccgg gcgaacccgg cgacaacgcg cgccgtcttg3540cggcgatctg cgcgggcgtc gtgctgtgtg gtgaattgtc gctgcttgcg gcccagacca3600accccggaga gttggtccgc acccacatgg agatggagcg atgaccgaca gcaaggatca3660ccatgtcgcg gggcgcaagc tggaccatct gcgtgcattg gacgacgatg cggatatcga3720ccggggcgac agcggcttcg accgcatcgc gctgacccat cgcgccctgc ccgaggtgga3780tttcgacgcc atcgacacgg cgaccagctt cctgggccgt gaactgtcct tcccgctgct3840gatctcgtcc atgaccggcg gcaccggcga ggagatcgag cgcatcaacc gcaacctggc3900cgctggtgcc gaggaggccc gcgtcgccat ggcggtgggc tcgcagcgcg tgatgttcac3960cgacccctcg gcgcgggcca gcttcgacct gcgcgcccat gcgcccaccg tgccgctgct4020ggccaatatc ggcgcggtgc agctgaacat ggggctgggg ctgaaggaat gcctggccgc4080gatcgaggtg ctgcaggcgg acggcctgta tctgcacctg aaccccctgc aagaggccgt4140
ccagcccgag ggggatcgcg actttgccga tctgggcagc aagatcgcgg ccatcgcccg4200cgacgttccc gtgcccgtcc tgctgaagga ggtgggctgc ggcctgtcgg cggccgatat4260cgccatcggg ctgcgcgccg ggatccggca tttcgacgtg gccggtcgcg gcggcacatc4320ctggagccgg atcgagtatc gccgccgcca gcgggccgat gacgacctgg gcctggtctt4380ccaggactgg ggcctgcaga ccgtggacgc cctgcgcgag gcgcggcccg cgcttgcggc4440ccatgatgga accagcgtgc tgatcgccag cggcggcatc cgcaacggtg tcgacatggc4500gaaatgcgtc atcctggggg ccgacatgtg cggggtcgcc gcgcccctgc tgaaagcggc4560ccaaaactcg cgcgaggcgg ttgtatccgc catccggaaa ctgcatctgg agttccggac4620agccatgttc ctcctgggtt gcggcacgct tgccgacctg aaggacaatt cctcgcttat4680ccgtcaatga aagtgcctaa gatgaccgtg acaggaatcg aagcgatcag cttctacacc4740ccccagaact acgtgggact ggatatcctt gccgcgcatc acgggatcga ccccgagaag4800ttctcgaagg ggatcgggca ggagaaaatc gcactgcccg gccatgacga ggatatcgtg4860accatggccg ccgaggccgc gctgccgatc atcgaacgcg cgggcacgca gggcatcgac4920acggttctgt tcgccaccga gagcgggatc gaccagtcga aggccgccgc catctatctg4980cgccgcctgc tggacctgtc gcccaactgc cgttgcgtcg agctgaagca ggcctgctat5040tccgcgacgg cggcgctgca gatggcctgc gcgcatgtcg cccgcaagcc cgaccgcaag5100gtgctggtga tcgcgtccga tgtcgcgcgc tatgaccgcg aaagctcggg cgaggcgacg5160cagggtgcgg gcgccgtcgc catccttgtc agcgccgatc ccaaggtggc cgagatcggc5220accgtctcgg ggctgttcac cgaggatatc atggatttct ggcggccgaa ccaccgccgc5280acgcccctgt tcgacggcaa ggcatcgacg ctgcgctatc tgaacgcgct ggtcgaggcg5340tggaacgact atcgcgcgaa tggcggccac gagttcgccg atttcgcgca tttctgctat5400cacgtgccgt tctcgcggat gggcgagaag gcgaacagcc acctggccaa ggcgaacaag5460acgccggtgg acatggggca ggtgcagacg ggcctgatct acaaccggca ggtcgggaac5520tgctataccg ggtcgatcta cctggcattc gcctcgctgc tggagaacgc tcaggaggac5580ctgaccggcg cgctggtcgg tctgttcagc tatggctcgg gtgcgacggg cgaattcttc5640gatgcgcgga tcgcgcccgg ttaccgcgac cacctgttcg cggaacgcca tcgcgaattg5700ctgcaggatc gcacgcccgt cacatatgac gaatacgttg ccctgtggga cgagatcgac5760ctgacgcagg gcgcgcccga caaggcgcgc ggtcgtttca ggctggcagg tatcgaggac5820gagaagcgca tct atg tcg acc ggc agg cct gaa gca ggc gcc cat gcc 5869Met Ser Thr Gly Arg Pro Glu Ala Gly Ala His Ala1 5 10ccg ggc aag ctg atc ctg tcc ggg gaa cat tcc gtg ctc tat ggt gcg 5917Pro Gly Lys Leu Ile Leu Ser Gly Glu His Ser Val Leu Tyr Gly Ala15 20 25ccc gcg ctt gcc atg gcc atc gcc cgc tat acc gag gtg tgg ttc acg 5965Pro Ala Leu Ala Met Ala Ile Ala Arg Tyr Thr Glu Val Trp Phe Thr30 35 40ccg ctt ggc att ggc gag ggg ata cgc acg aca ttc gcc aat ctc tcg 6013Pro Leu Gly Ile Gly Glu Gly Ile Arg Thr Thr Phe Ala Asn Leu Ser45 50 55 60ggc ggg gcg acc tat tcg ctg aag ctg ctg tcg ggg ttc aag tcg cgg 6061Gly Gly Ala Thr Tyr Ser Leu Lys Leu Leu Ser Gly Phe Lys Ser Arg65 70 75ctg gac cgc cgg ttc gag cag ttc ctg aac ggc gac cta aag gtg cac 6109Leu Asp Arg Arg Phe Glu Gln Phe Leu Asn Gly Asp Leu Lys Val His80 85 90aag gtc ctg acc cat ccc gac gat ctg gcg gtc tat gcg ctg gcg tcg 6157Lys Val Leu Thr His Pro Asp Asp Leu Ala Val Tyr Ala Leu Ala Ser95 100 105ctt ctg cac gac aag ccg ccg ggg acc gcc gcg atg ccg ggc atc ggc 6205Leu Leu His Asp Lys Pro Pro Gly Thr Ala Ala Met Pro Gly Ile Gly110 115 120
gcg atg cac cac ctg ccg cga ccg ggt gag ctg ggc agc cgg acg gag 6253Ala Met His His Leu Pro Arg Pro Gly Glu Leu Gly Ser Arg Thr Glu125 130 135 140ctg ccc atc ggc gcg ggc atg ggg tcg tct gcg gcc atc gtc gcg gcc 6301Leu Pro Ile Gly Ala Gly Met Gly Ser Ser Ala Ala Ile Val Ala Ala145 150 155acc acg gtc ctg ttc gag acg ctg ctg gac cgg ccc aag acg ccc gaa 6349Thr Thr Val Leu Phe Glu Thr Leu Leu Asp Arg Pro Lys Thr Pro Glu160 165 170cag cgc ttc gac cgc gtc cgc ttc tgc gag cgg ttg aag cac ggc aag 6397Gln Arg Phe Asp Arg Val Arg Phe Cys Glu Arg Leu Lys His Gly Lys175 180 185gcc ggt ccc atc gac gcg gcc agc gtc gtg cgc ggc ggg ctt gtc cgc 6445Ala Gly Pro Ile Asp Ala Ala Ser Val Val Arg Gly Gly Leu Val Arg190 195 200gtg ggc ggg aac ggg ccg ggt tcg atc agc agc ttc gat ttg ccc gag 6493Val Gly Gly Asn Gly Pro Gly Ser Ile Ser Ser Phe Asp Leu Pro Glu205 210 215 220gat cac gac ctt gtc gcg gga cgc ggc tgg tac tgg gta ctg cac ggg 6541Asp His Asp Leu Val Ala Gly Arg Gly Trp Tyr Trp Val Leu His Gly225 230 235cgc ccc gtc agc ggg acc ggc gaa tgc gtc agc gcg gtc gcg gcg gcg 6589Arg Pro Val Ser Gly Thr Gly Glu Cys Val Ser Ala Val Ala Ala Ala240 245 250cat ggt cgc gat gcg gcg ctg tgg gac gcc ttc gca gtc tgc acc cgc 6637His Gly Arg Asp Ala Ala Leu Trp Asp Ala Phe Ala Val Cys Thr Arg255 260 265gcg ttg gag gcc gcg ctg ctg tct ggg ggc agc ccc gac gcc gcc atc 6685Ala Leu Glu Ala Ala Leu Leu Ser Gly Gly Ser Pro Asp Ala Ala Ile270 275 280acc gag aac cag cgc ctg ctg gaa cgc atc ggc gtc gtg ccg gca gcg 6733Thr Glu Asn Gln Arg Leu Leu Glu Arg Ile Gly Val Val Pro Ala Ala285 290 295 300acg cag gcc ctc gtg gcc cag atc gag gag gcg ggt ggc gcg gcc aag 6781Thr Gln Ala Leu Val Ala Gln Ile Glu Glu Ala Gly Gly Ala Ala Lys305 310 315atc tgc ggc gca ggt tcc gtg cgg ggc gat cac ggc ggg gcg gtc ctc 6829Ile Cys Gly Ala Gly Ser Val Arg Gly Asp His Gly Gly Ala Val Leu320 325 330gtg cgg att gac gac gcg cag gcg atg gct tcg gtc atg gcg cgc cat 6877Val Arg Ile Asp Asp Ala Gln Ala Met Ala Ser Val Met Ala Arg His335 340 345ccc gac ctc gac tgg gcg ccc ctg cgc atg tcg cgc acg ggg gcg gca 6925Pro Asp Leu Asp Trp Ala Pro Leu Arg Met Ser Arg Thr Gly Ala Ala350 355 360
ccc ggc ccc gcg ccg cgt gcg caa ccg ctg ccg ggg cag ggc tga 6970Pro Gly Pro Ala Pro Arg Ala Gln Pro Leu Pro Gly Gln Gly365 370 375tggatcaggt catccgcgcc agcgcgccgg gttcggtcat gatcacgggc gaacatgccg7030tggtctatgg acaccgcgcc atcgtcgccg ggatcgagca gcgcgcccat gtgacgatcg7090tcccgcgtgc cgaccgcatg tttcgcatca cctcgcagat cggggcgccg cagcaggggt7150cgctggacga tctgcctgcg ggcgggacct atcgcttcgt gctggccgcc atcgcgcgac7210acgcgccgga cctgccttgc gggttcgaca tggacatcac ctcggggatc gatccgaggc7270tcgggcttgg atcctcggcg gcggtgacgg tcgcctgcct cggcgcgctg tcgcggctgg7330cggggcgggg gaccgagggg ctgcatgacg acgcgctgcg catcgtccgc gccatccagg7390gcaggggcag cggggccgat ctggcggcca gcctgcatgg cggcttcgtc gcctatcgcg7450cgcccgatgg cggtgccgcg cagatcgagg cgcttccggt gccgccgggg ccgttcggcc7510tgcgctatgc gggctacaag accccgacag ccgaggtgct gcgccttgtg gccgatcgga7570tggcgggcaa cgaggccgct ttcgacgcgc tctactcccg gatgggcgca agcgcagatg7630ccgcgatccg cgcggcgcaa gggctggact gggctgcatt ccacgacgcg ctgaacgaat7690accagcgcct gatggagcag ctgggcgtgt ccgacgacac gctggacgcg atcatccgcg7750aggcgcgcga cgcgggcgcc gcagtcgcca agatctccgg ctcggggctg ggggattgcg7810tgctggcact gggcgaccag cccaagggtt tcgtgcccgc aagcattgcc gagaagggac7870ttgttttcga tgactgatgc cgtccgcgac atgatcgccc gtgccatggc gggcgcgacc7930gacatccgag cagccgaggc ttatgcgccc agcaacatcg cgctgtcgaa atactggggc7990aagcgcgacg ccgcgcggaa ccttccgctg aacagctccg tctcgatctc gttggcgaac8050tggggctctc atacgcgggt cgaggggtcc ggcacgggcc acgacgaggt gcatcacaac8110ggcacgctgc tggatccggg cgacgccttc gcgcgccgcg cgttggcatt cgctgacctg8170ttccgggggg ggaggcacct gccgctgcgg atcacgacgc agaactcgat cccgacggcg8230gcggggcttg cctcgtcggc ctcggggttc gcggcgctga cccgtgcgct ggcgggggcg8290ttcgggctgg atctggacga cacggatctg agccgcatcg cccggatcgg cagtggcagc8350gccgcccgct cgatctggca cggcttcgtc cgctggaacc ggggcgaggc cgaggatggg8410catgacagcc acggcgtccc gctggacctg cgctggcccg gcttccgcat cgcgatcgtg8470gccgtggaca aggggcccaa gcctttcagt tcgcgcgacg gcatgaacca cacggtcgag8530accagcccgc tgttcccgcc ctggcctgcg caggcggaag cggattgccg cgtcatcgag8590gatgcgatcg ccgcccgcga catggccgcc ctgggtccgc gggtcgaggc gaacgccctt8650gcgatgcacg ccacgatgat ggccgcgcgc ccgccgctct gctacctgac gggcggcagc8710tggcaggtgc tggaacgcct gtggcaggcc cgcgcggacg ggcttgcggc ctttgcgacg8770atggatgccg gcccgaacgt caagctgatc ttcgaggaaa gcagcgccgc cgacgtgctg8830tacctgttcc ccgacgccag cctgatcgcg ccgttcgagg ggcgttgaac gcgtaagacg8890accactgggt aaggttctgc cgcgcgtggt ctcgactgcc tgcaaagagg tgcttgagtt8950gctgcgtgac tgcggcggcc gacttcgtgg gacttgcccg ccacgctgac gcgctggaaa9010cgcgcccgcg gattacgacc gcgtcattgc cctgaaccaa tttcccgtcg gtcgac9066<210>49<211>378<212>PRT<213>副球菌属菌种(Paracoccus sp.)<400>49Met Ser Thr Gly Arg Pro Glu Ala Gly Ala His Ala Pro Gly Lys Leu1 5 10 15Ile Leu Ser Gly Glu His Ser Val Leu Tyr Gly Ala Pro Ala Leu Ala20 25 30Met Ala Ile Ala Arg Tyr Thr Glu Val Trp Phe Thr Pro Leu Gly Ile35 40 45Gly Glu Gly Ile Arg Thr Thr Phe Ala Asn Leu Ser Gly Gly Ala Thr50 55 60Tyr Ser Leu Lys Leu Leu Ser Gly Phe Lys Ser Arg Leu Asp Arg Arg65 70 75 80
Phe Glu Gln Phe Leu Ash Gly Asp Leu Lys Val His Lys Val Leu Thr85 90 95His Pro Asp Asp Leu Ala Val Tyr Ala Leu Ala Ser Leu Leu His Asp100 105 110Lys Pro Pro Gly Thr Ala Ala Met Pro Gly Ile Gly Ala Met His His115 120 125Leu Pro Arg Pro Gly Glu Leu Gly Ser Arg Thr Glu Leu Pro Ile Gly130 135 140Ala Gly Met Gly Ser Ser Ala Ala Ile Val Ala Ala Thr Thr Val Leu145 150 155 160Phe Glu Thr Leu Leu Asp Arg Pro Lys Thr Pro Glu Gln Arg Phe Asp165 170 175Arg Val Arg Phe Cys Glu Arg Leu Lys His Gly Lys Ala Gly Pro Ile180 185 190Asp Ala Ala Ser Val Val Arg Gly Gly Leu Val Arg Val Gly Gly Asn195 200 205Gly Pro Gly Ser Ile Ser Ser Phe Asp Leu Pro Glu Asp His Asp Leu210 215 220Val Ala Gly Arg Gly Trp Tyr Trp Val Leu His Gly Arg Pro Val Ser225 230 235 240Gly Thr Gly Glu Cys Val Ser Ala Val Ala Ala Ala His Gly Arg Asp245 250 255Ala Ala Leu Trp Asp Ala Phe Ala Val Cys Thr Arg Ala Leu Glu Ala260 265 270Ala Leu Leu Ser Gly Gly Ser Pro Asp Ala Ala Ile Thr Glu Asn Gln275 280 285Arg Leu Leu Glu Arg Ile Gly Val Val Pro Ala Ala Thr Gln Ala Leu290 295 300Val Ala Gln Ile Glu Glu Ala Gly Gly Ala Ala Lys Ile Cys Gly Ala305 310 315 320Gly Ser Val Arg Gly Asp His Gly Gly Ala Val Leu Val Arg Ile Asp325 330 335Asp Ala Gln Ala Met Ala Ser Val Met Ala Arg His Pro Asp Leu Asp340 345 350Trp Ala Pro Leu Arg Met Ser Arg Thr Gly Ala Ala Pro Gly Pro Ala355 360 365Pro Arg Ala Gln Pro Leu Pro Gly Gln Gly370 375<210>50<211>9066
<212>DNA<213>副球菌属菌种(Paracoccus sp.)<220>
<221>CDS<222>(6970)..(7887)<223>
<400>50ggatccggca gctcgacacg ccgcagaacc tgtacgaacg tcccgccagc cgcttcgtcg 60cggaattcgt cgggcgcggg acggtggtgc ccgtgcaggc ccatgacggc gcgggccgcg 120cccgcatcct gggggccgag gtggcggtga acgccgcccc gcaatcgcgc tttgtcgatc 180acgtctgcct gcgccccgag aaccttgcca tctccgagac gggcgacctg cgcgccaagg 240tcgcgcgcgt cacctatctt ggcgggaaat acctgctgga aaccgtgctg gattgcggca 300cccggctggt gaccgagacc cgcgcccgct tcgatacggg cgcgcagctt ggcctgacca 360tcaacgcccc ctgggccttt gccgaggatt gaatggacag cgtgaagatc ctttcgggca 420tgggcgtgaa gggccctgcc tgcatcaggc tggatgtcgg cgggatgcgc ctgatcctcg 480attgcgggac cggcccggac gagggcgcgg agttcgaccc cgcctggctg gcggacgcgg 540atgcggtgct gatcacccat gaccacgtgg accatatcgg cggcgcgcgt cacgcggtcg 600cggcggggct gccgatccat gcgacgcggc agacggcggg gttgctgccc gcgggggcgg 660atctgcgcct gctgcccgaa cgcggtgtca cgcggatcgc cggggtcgat ctgacgaccg 720gtcgcaacgg gcatgccgcg ggcggcgtct ggatgcattt cgacatgggc gaggggctgt 780tctattccgg cgactggtcc gaggaatccg actggttcgc cttcgatccg cccccgcctg 840cggggacggc gattctcgac tgctcctatg gcggtttcga cgtggcgcaa tcggattgca 900tcgcggacct ggacgacctg ctcgaggtgc tgccggggca ggtactgctg ccggtgccgc 960catccggccg cgcggccgag ctggccctgc ggctgatccg ccgccacgga ccgggcagcg1020tgatggtcga cgacgcctgc ctgccggcca tcgcgcaact gcccgaggcg cgcggactgg1080cctacgccac cgaggcacgc tttcttgtct gcgacacgcc gaacgccgaa agccggcgcg1140gcatggcggc atctgcaagc atggcgcgat gcgggcaggc tggggcggga cgcgcatgtc1200gtcttcaccg ggcacatgaa cgtccatgcg cgcgcattct gcgaccgccc cggcgggcat1260ttccgccgct ggaacgtgca tccgccgctg cgcgaccagc gacggatgct ggaacggctg1320gccgcgcggc gctttgcccc ggccttctgc cccgaccccg agatctatct ggcgctggac1380atgggcgcgc aggtcttcat gcaccaggag gtgacgccat gatccccgcc cgcagcttct1440gcctgatccg ccacggcgaa acgaccgcca atgcaggggc gatcatcgcg ggcgcaaccg1500atgtgcccct gacgccaagg ggccgcgatc aggcccgcgc cctggcaggg cgcgaatggc1560catcgggcat cgcgctgttc gccagcccga tgtcgcgtgc ccgcgatacc gcgctgctgg1620cctttccggg gcgcgaccac cagcccgaac ccgatctgcg cgaacgcgac tggggcatct1680tcgagggacg ccccgtcgcc gatctgcccc cgcgcgaaat cacgccgcag gggggcgagg1740gctgggacga cgtgatggcc cgcgtggacc gcgcgatccg gcggatctgc gcgacctcgg1800gcgatgcgct gccggtgctg gtctgccatt cgggcgtgat ccgtgccgcg cgcgtgctgt1860ggaccaccgg cgatgcgggc gatcgtccgc ccaacgccac gccgatcctg ttcagcccgg1920acggcgaccg attaaaggaa ggaacgatat gaccgccacc accccctgcg tcgtcttcga1980acgtggacgg cacgcttgcc gaattcgacg ccgaccgcct gggccatctt gtccacggca2040cgaccaagca ctgggacgcc ttccaccacg cgatggccga cgccccgccc atccccgagg2100tcgcccgcct gatgcgcaag ctgaaggagg ggggcgagac ggtcgtcatc tgctcggggc2160ggccccgcgg ctggcaggat cagacgatcg catggctgcg caagcacgac ctgcccttcg2220acgggatcta tctgcgcccc gaggatcagg acggcgccag cgaccccgag gtcaagcgcc2280gcgccctagc cgagatgcgc gccgacgggc tggcgccctg gctggtcgtg gacgaccggc2340ggtccgtcgt ggatgcctgg cgggccgagg ggctggtctg cctgcaatgc gcgccggggg2400acttctaggg ccgcgcgacg ggggcgcgga caggctgggc gggaaaccgc cccgccacca2460tgtcctgcac gcgtcgaacc gcccgtccga cgccggtttc cgcacggaaa cgcgcggcaa2520gttgacataa cttgcacgcg acgtctcgat tctgcccgcg aagaatgcga tgcatccaga2580tgatgcagaa cgaagaagcg gaagcgcccg tgaaagacca gatgatttcc cataccccgg2640tgcccacgca atgggtcggc ccgatcctgt tccgcggccc cgtcgtcgag ggcccgatca2700gcgcgccgct ggccacctac gagacgccgc tctggccctc gaccgcgcgg ggggcagggg2760tttcccggca ttcgggcggg atccaggtct cgctggtcga cgaacgcatg agccgctcga2820tcgcgctgcg ggcgcatgac ggggcggcgg cgaccgccgc ctggcagtcg atcaaggccc2880gccaggaaga ggtcgcggcc gtggtcgcca ccaccagccg cttcgcccgc cttgtcgagc2940tgaatcgcca gatcgtgggc aacctgcttt acatccgcat cgaatgcgtg acgggcgacg3000cctcgggtca caacatggtc accaaggccg ccgaggccgt gcagggctgg atcctgtcgg3060aatacccgat gctggcctat tccacgatct cggggaacct gtgcaccgac aagaaggcgt3120cggcggtcaa cggcatcctg ggccgcggca aatacgccgt cgccgaggtc gagatcccgc3180
gcaagatcct gacccgcgtg ctgcgcacca gcgccgagaa gatggtccgc ctgaactacg3240agaagaacta tgtcgggggt acgctggcgg ggtcgctgcg cagtgcgaac gcgcatttcg3300ccaacatgct gctgggcttc tacctggcga cggggcagga cgcggccaac atcatcgagg3360ccagccaggg cttcgtccat tgcgaggccc gcggcgagga tctgtatttc tcgtgcacgc3420tgcccaacct catcatgggc tcggtcggtg ccggcaaggg catcccctcg atcgaggaga3480acctgtcgcg gatgggctgc cgccagccgg gcgaacccgg cgacaacgcg cgccgtcttg3540cggcgatctg cgcgggcgtc gtgctgtgtg gtgaattgtc gctgcttgcg gcccagacca3600accccggaga gttggtccgc acccacatgg agatggagcg atgaccgaca gcaaggatca3660ccatgtcgcg gggcgcaagc tggaccatct gcgtgcattg gacgacgatg cggatatcga3720ccggggcgac agcggcttcg accgcatcgc gctgacccat cgcgccctgc ccgaggtgga3780tttcgacgcc atcgacacgg cgaccagctt cctgggccgt gaactgtcct tcccgctgct3840gatctcgtcc atgaccggcg gcaccggcga ggagatcgag cgcatcaacc gcaacctggc3900cgctggtgcc gaggaggccc gcgtcgccat ggcggtgggc tcgcagcgcg tgatgttcac3960cgacccctcg gcgcgggcca gcttcgacct gcgcgcccat gcgcccaccg tgccgctgct4020ggccaatatc ggcgcggtgc agctgaacat ggggctgggg ctgaaggaat gcctggccgc4080gatcgaggtg ctgcaggcgg acggcctgta tctgcacctg aaccccctgc aagaggccgt4140ccagcccgag ggggatcgcg actttgccga tctgggcagc aagatcgcgg ccatcgcccg4200cgacgttccc gtgcccgtcc tgctgaagga ggtgggctgc ggcctgtcgg cggccgatat4260cgccatcggg ctgcgcgccg ggatccggca tttcgacgtg gccggtcgcg gcggcacatc4320ctggagccgg atcgagtatc gccgccgcca gcgggccgat gacgacctgg gcctggtctt4380ccaggactgg ggcctgcaga ccgtggacgc cctgcgcgag gcgcggcccg cgcttgcggc4440ccatgatgga accagcgtgc tgatcgccag cggcggcatc cgcaacggtg tcgacatggc4500gaaatgcgtc atcctggggg ccgacatgtg cggggtcgcc gcgcccctgc tgaaagcggc4560ccaaaactcg cgcgaggcgg ttgtatccgc catccggaaa ctgcatctgg agttccggac4620agccatgttc ctcctgggtt gcggcacgct tgccgacctg aaggacaatt cctcgcttat4680ccgtcaatga aagtgcctaa gatgaccgtg acaggaatcg aagcgatcag cttctacacc4740ccccagaact acgtgggact ggatatcctt gccgcgcatc acgggatcga ccccgagaag4800ttctcgaagg ggatcgggca ggagaaaatc gcactgcccg gccatgacga ggatatcgtg4860accatggccg ccgaggccgc gctgccgatc atcgaacgcg cgggcacgca gggcatcgac4920acggttctgt tcgccaccga gagcgggatc gaccagtcga aggccgccgc catctatctg4980cgccgcctgc tggacctgtc gcccaactgc cgttgcgtcg agctgaagca ggcctgctat5040tccgcgacgg cggcgctgca gatggcctgc gcgcatgtcg cccgcaagcc cgaccgcaag5100gtgctggtga tcgcgtccga tgtcgcgcgc tatgaccgcg aaagctcggg cgaggcgacg5160cagggtgcgg gcgccgtcgc catccttgtc agcgccgatc ccaaggtggc cgagatcggc5220accgtctcgg ggctgttcac cgaggatatc atggatttct ggcggccgaa ccaccgccgc5280acgcccctgt tcgacggcaa ggcatcgacg ctgcgctatc tgaacgcgct ggtcgaggcg5340tggaacgact atcgcgcgaa tggcggccac gagttcgccg atttcgcgca tttctgctat5400cacgtgccgt tctcgcggat gggcgagaag gcgaacagcc acctggccaa ggcgaacaag5460acgccggtgg acatggggca ggtgcagacg ggcctgatct acaaccggca ggtcgggaac5520tgctataccg ggtcgatcta cctggcattc gcctcgctgc tggagaacgc tcaggaggac5580ctgaccggcg cgctggtcgg tctgttcagc tatggctcgg gtgcgacggg cgaattcttc5640gatgcgcgga tcgcgcccgg ttaccgcgac cacctgttcg cggaacgcca tcgcgaattg5700ctgcaggatc gcacgcccgt cacatatgac gaatacgttg ccctgtggga cgagatcgac5760ctgacgcagg gcgcgcccga caaggcgcgc ggtcgtttca ggctggcagg tatcgaggac5820gagaagcgca tctatgtcga ccggcaggcc tgaagcaggc gcccatgccc cgggcaagct5880gatcctgtcc ggggaacatt ccgtgctcta tggtgcgccc gcgcttgcca tggccatcgc5940ccgctatacc gaggtgtggt tcacgccgct tggcattggc gaggggatac gcacgacatt6000cgccaatctc tcgggcgggg cgacctattc gctgaagctg ctgtcggggt tcaagtcgcg6060gctggaccgc cggttcgagc agttcctgaa cggcgaccta aaggtgcaca aggtcctgac6120ccatcccgac gatctggcgg tctatgcgct ggcgtcgctt ctgcacgaca agccgccggg6180gaccgccgcg atgccgggca tcggcgcgat gcaccacctg ccgcgaccgg gtgagctggg6240cagccggacg gagctgccca tcggcgcggg catggggtcg tctgcggcca tcgtcgcggc6300caccacggtc ctgttcgaga cgctgctgga ccggcccaag acgcccgaac agcgcttcga6360ccgcgtccgc ttctgcgagc ggttgaagca cggcaaggcc ggtcccatcg acgcggccag6420cgtcgtgcgc ggcgggcttg tccgcgtggg cgggaacggg ccgggttcga tcagcagctt6480cgatttgccc gaggatcacg accttgtcgc gggacgcggc tggtactggg tactgcacgg6540gcgccccgtc agcgggaccg gcgaatgcgt cagcgcggtc gcggcggcgc atggtcgcga6600tgcggcgctg tgggacgcct tcgcagtctg cacccgcgcg ttggaggccg cgctgctgtc6660tgggggcagc cccgacgccg ccatcaccga gaaccagcgc ctgctggaac gcatcggcgt6720cgtgccggca gcgacgcagg ccctcgtggc ccagatcgag gaggcgggtg gcgcggccaa6780
gatctgcggc gcaggttccg tgcggggcga tcacggcggg gcggtcctcg tgcggattga6840cgacgcgcag gcgatggctt cggtcatggc gcgccatccc gacctcgact gggcgcccct6900gcgcatgtcg cgcacggggg cggcacccgg ccccgcgccg cgtgcgcaac cgctgccggg6960gcagggctg atg gat cag gtc atc cgc gcc agc gcg ccg ggt tcg gtc atg7011Met Asp Gln Val Ile Arg Ala Ser Ala Pro Gly Ser Val Met1 5 10atc acg ggc gaa cat gcc gtg gtc tat gga cac cgc gcc atc gtc gcc 7059Ile Thr Gly Glu His Ala Val Val Tyr Gly His Arg Ala Ile Val Ala15 20 25 30ggg atc gag cag cgc gcc cat gtg acg atc gtc ccg cgt gcc gac cgc 7107Gly Ile Glu Gln Arg Ala His Val Thr Ile Val Pro Arg Ala Asp Arg35 40 45atg ttt cgc atc acc tcg cag atc ggg gcg ccg cag cag ggg tcg ctg 7155Met Phe Arg Ile Thr Ser Gln Ile Gly Ala Pro Gln Gln Gly Ser Leu50 55 60gac gat ctg cct gcg ggc ggg acc tat cgc ttc gtg ctg gcc gcc atc 7203Asp Asp Leu Pro Ala Gly Gly Thr Tyr Arg Phe Val Leu Ala Ala Ile65 70 75gcg cga cac gcg ccg gac ctg cct tgc ggg ttc gac atg gac atc acc 7251Ala Arg His Ala Pro Asp Leu Pro Cys Gly Phe Asp Met Asp Ile Thr80 85 90tcg ggg atc gat ccg agg ctc ggg ctt gga tcc tcg gcg gcg gtg acg 7299Ser Gly Ile Asp Pro Arg Leu Gly Leu Gly Ser Ser Ala Ala Val Thr95 100 105 110gtc gcc tgc ctc ggc gcg ctg tcg cgg ctg gcg ggg cgg ggg acc gag 7347Val Ala Cys Leu Gly Ala Leu Ser Arg Leu Ala Gly Arg Gly Thr Glu115 120 125ggg ctg cat gac gac gcg ctg cgc atc gtc cgc gcc atc cag ggc agg 7395Gly Leu His Asp Asp Ala Leu Arg Ile Val Arg Ala Ile Gln Gly Arg130 135 140ggc agc ggg gcc gat ctg gcg gcc agc ctg cat ggc ggc ttc gtc gcc 7443Gly Ser Gly Ala Asp Leu Ala Ala Ser Leu His Gly Gly Phe Val Ala145 150 155tat cgc gcg ccc gat ggc ggt gcc gcg cag atc gag gcg ctt ccg gtg 7491Tyr Arg Ala Pro Asp Gly Gly Ala Ala Gln Ile Glu Ala Leu Pro Val160 165 170ccg ccg ggg ccg ttc ggc ctg cgc tat gcg ggc tac aag acc ccg aca 7539Pro Pro Gly Pro Phe Gly Leu Arg Tyr Ala Gly Tyr Lys Thr Pro Thr175 180 185 190gcc gag gtg ctg cgc ctt gtg gcc gat cgg atg gcg ggc aac gag gcc 7587Ala Glu Val Leu Arg Leu Val Ala Asp Arg Met Ala Gly Asn Glu Ala195 200 205gct ttc gac gcg ctc tac tcc cgg atg ggc gca agc gca gat gcc gcg 7635Ala Phe Asp Ala Leu Tyr Ser Arg Met Gly Ala Ser Ala Asp Ala Ala210 215 220atc cgc gcg gcg caa ggg ctg gac tgg gct gca ttc cac gac gcg ctg 7683Ile Arg Ala Ala Gln Gly Leu Asp Trp Ala Ala Phe His Asp Ala Leu
225 230 235aac gaa tac cag cgc ctg atg gag cag ctg ggc gtg tcc gac gac acg 7731Asn Glu Tyr Gln Arg Leu Met Glu Gln Leu Gly Val Ser Asp Asp Thr240 245 250ctg gac gcg atc atc cgc gag gcg cgc gac gcg ggc gcc gca gtc gcc 7779Leu Asp Ala Ile Ile Arg Glu Ala Arg Asp Ala Gly Ala Ala Val Ala255 260 265 270aag atc tcc ggc tcg ggg ctg ggg gat tgc gtg ctg gca ctg ggc gac 7827Lys Ile Ser Gly Ser Gly Leu Gly Asp Cys Val Leu Ala Leu Gly Asp275 280 285cag ccc aag ggt ttc gtg ccc gca agc att gcc gag aag gga ctt gtt 7875Gln Pro Lys Gly Phe Val Pro Ala Ser Ile Ala Glu Lys Gly Leu Val290 295 300ttc gat gac tga tgccgtccgc gacatgatcg cccgtgccat ggcgggcgcg 7927Phe Asp Asp305accgacatcc gagcagccga ggcttatgcg cccagcaaca tcgcgctgtc gaaatactgg7987ggcaagcgcg acgccgcgcg gaaccttccg ctgaacagct ccgtctcgat ctcgttggcg8047aactggggct ctcatacgcg ggtcgagggg tccggcacgg gccacgacga ggtgcatcac8107aacggcacgc tgctggatcc gggcgacgcc ttcgcgcgcc gcgcgttggc attcgctgac8167ctgttccggg gggggaggca cctgccgctg cggatcacga cgcagaactc gatcccgacg8227gcggcggggc ttgcctcgtc ggcctcgggg ttcgcggcgc tgacccgtgc gctggcgggg8287gcgttcgggc tggatctgga cgacacggat ctgagccgca tcgcccggat cggcagtggc8347agcgccgccc gctcgatctg gcacggcttc gtccgctgga accggggcga ggccgaggat8407gggcatgaca gccacggcgt cccgctggac ctgcgctggc ccggcttccg catcgcgatc8467gtggccgtgg acaaggggcc caagcctttc agttcgcgcg acggcatgaa ccacacggtc8527gagaccagcc cgctgttccc gccctggcct gcgcaggcgg aagcggattg ccgcgtcatc8587gaggatgcga tcgccgcccg cgacatggcc gccctgggtc cgcgggtcga ggcgaacgcc8647cttgcgatgc acgccacgat gatggccgcg cgcccgccgc tctgctacct gacgggcggc8707agctggcagg tgctggaacg cctgtggcag gcccgcgcgg acgggcttgc ggcctttgcg8767acgatggatg ccggcccgaa cgtcaagctg atcttcgagg aaagcagcgc cgccgacgtg8827ctgtacctgt tccccgacgc cagcctgatc gcgccgttcg aggggcgttg aacgcgtaag8887acgaccactg ggtaaggttc tgccgcgcgt ggtctcgact gcctgcaaag aggtgcttga8947gttgctgcgt gactgcggcg gccgacttcg tgggacttgc ccgccacgct gacgcgctgg9007aaacgcgccc gcggattacg accgcgtcat tgccctgaac caatttcccg tcggtcgac 9066<210>51<211>305<212>PRT<213>副球菌属菌种(Paracoccus sp.)<400>51Met Asp Gln Val Ile Arg Ala Ser Ala Pro Gly Ser Val Met Ile Thr1 5 10 15Gly Glu His Ala Val Val Tyr Gly His Arg Ala Ile Val Ala Gly Ile20 25 30Glu Gln Arg Ala His Val Thr Ile Val Pro Arg Ala Asp Arg Met Phe35 40 45Arg Ile Thr Ser Gln Ile Gly Ala Pro Gln Gln Gly Ser Leu Asp Asp50 55 60Leu Pro Ala Gly Gly Thr Tyr Arg Phe Val Leu Ala Ala Ile Ala Arg
65 70 75 80His Ala Pro Asp Leu Pro Cys Gly Phe Asp Met Asp Ile Thr Ser Gly85 90 95Ile Asp Pro Arg Leu Gly Leu Gly Ser Ser Ala Ala Val Thr Val Ala100 105 110Cys Leu Gly Ala Leu Ser Arg Leu Ala Gly Arg Gly Thr Glu Gly Leu115 120 125His Asp Asp Ala Leu Arg Ile Val Arg Ala Ile Gln Gly Arg Gly Ser130 135 140Gly Ala Asp Leu Ala Ala Ser Leu His Gly Gly Phe Val Ala Tyr Arg145 150 155 160Ala Pro Asp Gly Gly Ala Ala Gln Ile Glu Ala Leu Pro Val Pro Pro165 170 175Gly Pro Phe Gly Leu Arg Tyr Ala Gly Tyr Lys Thr Pro Thr Ala Glu180 185 190Val Leu Arg Leu Val Ala Asp Arg Met Ala Gly Asn Glu Ala Ala Phe195 200 205Asp Ala Leu Tyr Ser Arg Met Gly Ala Ser Ala Asp Ala Ala Ile Arg210 215 220Ala Ala Gln Gly Leu Asp Trp Ala Ala Phe His Asp Ala Leu Asn Glu225 230 235 240Tyr Gln Arg Leu Met Glu Gln Leu Gly Val Ser Asp Asp Thr Leu Asp245 250 255Ala Ile Ile Arg Glu Ala Arg Asp Ala Gly Ala Ala Val Ala Lys Ile260 265 270Ser Gly Ser Gly Leu Gly Asp Cys Val Leu Ala Leu Gly Asp Gln Pro275 280 285Lys Gly Phe Val Pro Ala Ser Ile Ala Glu Lys Gly Leu Val Phe Asp290 295 300Asp305<210>52<211>9066<212>DNA<213>副球菌属菌种(Paracoccus sp.)<220>
<221>CDS<222>(7880)..(8878)<223>
<400>52ggatccggca gctcgacacg ccgcagaacc tgtacgaacg tcccgccagc cgcttcgtcg 60cggaattcgt cgggcgcggg acggtggtgc ccgtgcaggc ccatgacggc gcgggccgcg120cccgcatcct gggggccgag gtggcggtga acgccgcccc gcaatcgcgc tttgtcgatc180acgtctgcct gcgccccgag aaccttgcca tctccgagac gggcgacctg cgcgccaagg240
tcgcgcgcgt cacctatctt ggcgggaaat acctgctgga aaccgtgctg gattgcggca 300cccggctggt gaccgagacc cgcgcccgct tcgatacggg cgcgcagctt ggcctgacca 360tcaacgcccc ctgggccttt gccgaggatt gaatggacag cgtgaagatc ctttcgggca 420tgggcgtgaa gggccctgcc tgcatcaggc tggatgtcgg cgggatgcgc ctgatcctcg 480attgcgggac cggcccggac gagggcgcgg agttcgaccc cgcctggctg gcggacgcgg 540atgcggtgct gatcacccat gaccacgtgg accatatcgg cggcgcgcgt cacgcggtcg 600cggcggggct gccgatccat gcgacgcggc agacggcggg gttgctgccc gcgggggcgg 660atctgcgcct gctgcccgaa cgcggtgtca cgcggatcgc cggggtcgat ctgacgaccg 720gtcgcaacgg gcatgccgcg ggcggcgtct ggatgcattt cgacatgggc gaggggctgt 780tctattccgg cgactggtcc gaggaatccg actggttcgc cttcgatccg cccccgcctg 840cggggacggc gattctcgac tgctcctatg gcggtttcga cgtggcgcaa tcggattgca 900tcgcggacct ggacgacctg ctcgaggtgc tgccggggca ggtactgctg ccggtgccgc 960catccggccg cgcggccgag ctggccctgc ggctgatccg ccgccacgga ccgggcagcg1020tgatggtcga cgacgcctgc ctgccggcca tcgcgcaact gcccgaggcg cgcggactgg1080cctacgccac cgaggcacgc tttcttgtct gcgacacgcc gaacgccgaa agccggcgcg1140gcatggcggc atctgcaagc atggcgcgat gcgggcaggc tggggcggga cgcgcatgtc1200gtcttcaccg ggcacatgaa cgtccatgcg cgcgcattct gcgaccgccc cggcgggcat1260ttccgccgct ggaacgtgca tccgccgctg cgcgaccagc gacggatgct ggaacggctg1320gccgcgcggc gctttgcccc ggccttctgc cccgaccccg agatctatct ggcgctggac1380atgggcgcgc aggtcttcat gcaccaggag gtgacgccat gatccccgcc cgcagcttct1440gcctgatccg ccacggcgaa acgaccgcca atgcaggggc gatcatcgcg ggcgcaaccg1500atgtgcccct gacgccaagg ggccgcgatc aggcccgcgc cctggcaggg cgcgaatggc1560catcgggcat cgcgctgttc gccagcccga tgtcgcgtgc ccgcgatacc gcgctgctgg1620cctttccggg gcgcgaccac cagcccgaac ccgatctgcg cgaacgcgac tggggcatct1680tcgagggacg ccccgtcgcc gatctgcccc cgcgcgaaat cacgccgcag gggggcgagg1740gctgggacga cgtgatggcc cgcgtggacc gcgcgatccg gcggatctgc gcgacctcgg1800gcgatgcgct gccggtgctg gtctgccatt cgggcgtgat ccgtgccgcg cgcgtgctgt1860ggaccaccgg cgatgcgggc gatcgtccgc ccaacgccac gccgatcctg ttcagcccgg1920acggcgaccg attaaaggaa ggaacgatat gaccgccacc accccctgcg tcgtcttcga1980acgtggacgg cacgcttgcc gaattcgacg ccgaccgcct gggccatctt gtccacggca2040cgaccaagca ctgggacgcc ttccaccacg cgatggccga cgccccgccc atccccgagg2100tcgcccgcct gatgcgcaag ctgaaggagg ggggcgagac ggtcgtcatc tgctcggggc2160ggccccgcgg ctggcaggat cagacgatcg catggctgcg caagcacgac ctgcccttcg2220acgggatcta tctgcgcccc gaggatcagg acggcgccag cgaccccgag gtcaagcgcc2280gcgccctagc cgagatgcgc gccgacgggc tggcgccctg gctggtcgtg gacgaccggc2340ggtccgtcgt ggatgcctgg cgggccgagg ggctggtctg cctgcaatgc gcgccggggg2400acttctaggg ccgcgcgacg ggggcgcgga caggctgggc gggaaaccgc cccgccacca2460tgtcctgcac gcgtcgaacc gcccgtccga cgccggtttc cgcacggaaa cgcgcggcaa2520gttgacataa cttgcacgcg acgtctcgat tctgcccgcg aagaatgcga tgcatccaga2580tgatgcagaa cgaagaagcg gaagcgcccg tgaaagacca gatgatttcc cataccccgg2640tgcccacgca atgggtcggc ccgatcctgt tccgcggccc cgtcgtcgag ggcccgatca2700gcgcgccgct ggccacctac gagacgccgc tctggccctc gaccgcgcgg ggggcagggg2760tttcccggca ttcgggcggg atccaggtct cgctggtcga cgaacgcatg agccgctcga2820tcgcgctgcg ggcgcatgac ggggcggcgg cgaccgccgc ctggcagtcg atcaaggccc2880gccaggaaga ggtcgcggcc gtggtcgcca ccaccagccg cttcgcccgc cttgtcgagc2940tgaatcgcca gatcgtgggc aacctgcttt acatccgcat cgaatgcgtg acgggcgacg3000cctcgggtca caacatggtc accaaggccg ccgaggccgt gcagggctgg atcctgtcgg3060aatacccgat gctggcctat tccacgatct cggggaacct gtgcaccgac aagaaggcgt3120cggcggtcaa cggcatcctg ggccgcggca aatacgccgt cgccgaggtc gagatcccgc3180gcaagatcct gacccgcgtg ctgcgcacca gcgccgagaa gatggtccgc ctgaactacg3240agaagaacta tgtcgggggt acgctggcgg ggtcgctgcg cagtgcgaac gcgcatttcg3300ccaacatgct gctgggcttc tacctggcga cggggcagga cgcggccaac atcatcgagg3360ccagccaggg cttcgtccat tgcgaggccc gcggcgagga tctgtatttc tcgtgcacgc3420tgcccaacct catcatgggc tcggtcggtg ccggcaaggg catcccctcg atcgaggaga3480acctgtcgcg gatgggctgc cgccagccgg gcgaacccgg cgacaacgcg cgccgtcttg3540cggcgatctg cgcgggcgtc gtgctgtgtg gtgaattgtc gctgcttgcg gcccagacca3600accccggaga gttggtccgc acccacatgg agatggagcg atgaccgaca gcaaggatca3660ccatgtcgcg gggcgcaagc tggaccatct gcgtgcattg gacgacgatg cggatatcga3720ccggggcgac agcggcttcg accgcatcgc gctgacccat cgcgccctgc ccgaggtgga3780tttcgacgcc atcgacacgg cgaccagctt cctgggccgt gaactgtcct tcccgctgct3840
gatctcgtcc atgaccggcg gcaccggcga ggagatcgag cgcatcaacc gcaacctggc3900cgctggtgcc gaggaggccc gcgtcgccat ggcggtgggc tcgcagcgcg tgatgttcac3960cgacccctcg gcgcgggcca gcttcgacct gcgcgcccat gcgcccaccg tgccgctgct4020ggccaatatc ggcgcggtgc agctgaacat ggggctgggg ctgaaggaat gcctggccgc4080gatcgaggtg ctgcaggcgg acggcctgta tctgcacctg aaccccctgc aagaggccgt4140ccagcccgag ggggatcgcg actttgccga tctgggcagc aagatcgcgg ccatcgcccg4200cgacgttccc gtgcccgtcc tgctgaagga ggtgggctgc ggcctgtcgg cggccgatat4260cgccatcggg ctgcgcgccg ggatccggca tttcgacgtg gccggtcgcg gcggcacatc4320ctggagccgg atcgagtatc gccgccgcca gcgggccgat gacgacctgg gcctggtctt4380ccaggactgg ggcctgcaga ccgtggacgc cctgcgcgag gcgcggcccg cgcttgcggc4440ccatgatgga accagcgtgc tgatcgccag cggcggcatc cgcaacggtg tcgacatggc4500gaaatgcgtc atcctggggg ccgacatgtg cggggtcgcc gcgcccctgc tgaaagcggc4560ccaaaactcg cgcgaggcgg ttgtatccgc catccggaaa ctgcatctgg agttccggac4620agccatgttc ctcctgggtt gcggcacgct tgccgacctg aaggacaatt cctcgcttat4680ccgtcaatga aagtgcctaa gatgaccgtg acaggaatcg aagcgatcag cttctacacc4740ccccagaact acgtgggact ggatatcctt gccgcgcatc acgggatcga ccccgagaag4800ttctcgaagg ggatcgggca ggagaaaatc gcactgcccg gccatgacga ggatatcgtg4860accatggccg ccgaggccgc gctgccgatc atcgaacgcg cgggcacgca gggcatcgac4920acggttctgt tcgccaccga gagcgggatc gaccagtcga aggccgccgc catctatctg4980cgccgcctgc tggacctgtc gcccaactgc cgttgcgtcg agctgaagca ggcctgctat5040tccgcgacgg cggcgctgca gatggcctgc gcgcatgtcg cccgcaagcc cgaccgcaag5100gtgctggtga tcgcgtccga tgtcgcgcgc tatgaccgcg aaagctcggg cgaggcgacg5160cagggtgcgg gcgccgtcgc catccttgtc agcgccgatc ccaaggtggc cgagatcggc5220accgtctcgg ggctgttcac cgaggatatc atggatttct ggcggccgaa ccaccgccgc5280acgcccctgt tcgacggcaa ggcatcgacg ctgcgctatc tgaacgcgct ggtcgaggcg5340tggaacgact atcgcgcgaa tggcggccac gagttcgccg atttcgcgca tttctgctat5400cacgtgccgt tctcgcggat gggcgagaag gcgaacagcc acctggccaa ggcgaacaag5460acgccggtgg acatggggca ggtgcagacg ggcctgatct acaaccggca ggtcgggaac5520tgctataccg ggtcgatcta cctggcattc gcctcgctgc tggagaacgc tcaggaggac5580ctgaccggcg cgctggtcgg tctgttcagc tatggctcgg gtgcgacggg cgaattcttc5640gatgcgcgga tcgcgcccgg ttaccgcgac cacctgttcg cggaacgcca tcgcgaattg5700ctgcaggatc gcacgcccgt cacatatgac gaatacgttg ccctgtggga cgagatcgac5760ctgacgcagg gcgcgcccga caaggcgcgc ggtcgtttca ggctggcagg tatcgaggac5820gagaagcgca tctatgtcga ccggcaggcc tgaagcaggc gcccatgccc cgggcaagct5880gatcctgtcc ggggaacatt ccgtgctcta tggtgcgccc gcgcttgcca tggccatcgc5940ccgctatacc gaggtgtggt tcacgccgct tggcattggc gaggggatac gcacgacatt6000cgccaatctc tcgggcgggg cgacctattc gctgaagctg ctgtcggggt tcaagtcgcg6060gctggaccgc cggttcgagc agttcctgaa cggcgaccta aaggtgcaca aggtcctgac6120ccatcccgac gatctggcgg tctatgcgct ggcgtcgctt ctgcacgaca agccgccggg6180gaccgccgcg atgccgggca tcggcgcgat gcaccacctg ccgcgaccgg gtgagctggg6240cagccggacg gagctgccca tcggcgcggg catggggtcg tctgcggcca tcgtcgcggc6300caccacggtc ctgttcgaga cgctgctgga ccggcccaag acgcccgaac agcgcttcga6360ccgcgtccgc ttctgcgagc ggttgaagca cggcaaggcc ggtcccatcg acgcggccag6420cgtcgtgcgc ggcgggcttg tccgcgtggg cgggaacggg ccgggttcga tcagcagctt6480cgatttgccc gaggatcacg accttgtcgc gggacgcggc tggtactggg tactgcacgg6540gcgccccgtc agcgggaccg gcgaatgcgt cagcgcggtc gcggcggcgc atggtcgcga6600tgcggcgctg tgggacgcct tcgcagtctg cacccgcgcg ttggaggccg cgctgctgtc6660tgggggcagc cccgacgccg ccatcaccga gaaccagcgc ctgctggaac gcatcggcgt6720cgtgccggca gcgacgcagg ccctcgtggc ccagatcgag gaggcgggtg gcgcggccaa6780gatctgcggc gcaggttccg tgcggggcga tcacggcggg gcggtcctcg tgcggattga6840cgacgcgcag gcgatggctt cggtcatggc gcgccatccc gacctcgact gggcgcccct6900gcgcatgtcg cgcacggggg cggcacccgg ccccgcgccg cgtgcgcaac cgctgccggg6960gcagggctga tggatcaggt catccgcgcc agcgcgccgg gttcggtcat gatcacgggc7020gaacatgccg tggtctatgg acaccgcgcc atcgtcgccg ggatcgagca gcgcgcccat7080gtgacgatcg tcccgcgtgc cgaccgcatg tttcgcatca cctcgcagat cggggcgccg7140cagcaggggt cgctggacga tctgcctgcg ggcgggacct atcgcttcgt gctggccgcc7200atcgcgcgac acgcgccgga cctgccttgc gggttcgaca tggacatcac ctcggggatc7260gatccgaggc tcgggcttgg atcctcggcg gcggtgacgg tcgcctgcct cggcgcgctg7320tcgcggctgg cggggcgggg gaccgagggg ctgcatgacg acgcgctgcg catcgtccgc7380gccatccagg gcaggggcag cggggccgat ctggcggcca gcctgcatgg cggcttcgtc7440
gcctatcgcg cgcccgatgg cggtgccgcg cagatcgagg cgcttccggt gccgccgggg7500ccgttcggcc tgcgctatgc gggctacaag accccgacag ccgaggtgct gcgccttgtg7560gccgatcgga tggcgggcaa cgaggccgct ttcgacgcgc tctactcccg gatgggcgca7620agcgcagatg ccgcgatccg cgcggcgcaa gggctggact gggctgcatt ccacgacgcg7680ctgaacgaat accagcgcct gatggagcag ctgggcgtgt ccgacgacac gctggacgcg7740atcatccgcg aggcgcgcga cgcgggcgcc gcagtcgcca agatctccgg ctcggggctg7800ggggattgcg tgctggcact gggcgaccag cccaagggtt tcgtgcccgc aagcattgcc7860gagaagggac ttgttttcg atg act gat gcc gtc cgc gac atg atc gcc cgt 7912Met Thr Asp Ala Val Arg Asp Met Ile Ala Arg1 5 10gcc atg gcg ggc gcg acc gac atc cga gca gcc gag gct tat gcg ccc 7960Ala Met Ala Gly Ala Thr Asp Ile Arg Ala Ala Glu Ala Tyr Ala Pro15 20 25agc aac atc gcg ctg tcg aaa tac tgg ggc aag cgc gac gcc gcg cgg 8008Ser Asn Ile Ala Leu Ser Lys Tyr Trp Gly Lys Arg Asp Ala Ala Arg30 35 40aac ctt ccg ctg aac agc tcc gtc tcg atc tcg ttg gcg aac tgg ggc 8056Asn Leu Pro Leu Asn Ser Ser Val Ser Ile Ser Leu Ala Asn Trp Gly45 50 55tct cat acg cgg gtc gag ggg tcc ggc acg ggc cac gac gag gtg cat 8104Ser His Thr Arg Val Glu Gly Ser Gly Thr Gly His Asp Glu Val His60 65 70 75cac aac ggc acg ctg ctg gat ccg ggc gac gcc ttc gcg cgc cgc gcg 8152His Asn Gly Thr Leu Leu Asp Pro Gly Asp Ala Phe Ala Arg Arg Ala80 85 90ttg gca ttc gct gac ctg ttc cgg ggg ggg agg cac ctg ccg ctg cgg 8200Leu Ala Phe Ala Asp Leu Phe Arg Gly Gly Arg His Leu Pro Leu Arg95 100 105atc acg acg cag aac tcg atc ccg acg gcg gcg ggg ctt gcc tcg tcg 8248Ile Thr Thr Gln Asn Ser Ile Pro Thr Ala Ala Gly Leu Ala Ser Ser110 115 120gcc tcg ggg ttc gcg gcg ctg acc cgt gcg ctg gcg ggg gcg ttc ggg 8296Ala Ser Gly Phe Ala Ala Leu Thr Arg Ala Leu Ala Gly Ala Phe Gly125 130 135ctg gat ctg gac gac acg gat ctg agc cgc atc gcc cgg atc ggc agt 8344Leu Asp Leu Asp Asp Thr Asp Leu Ser Arg Ile Ala Arg Ile Gly Ser140 145 150 155ggc agc gcc gcc cgc tcg atc tgg cac ggc ttc gtc cgc tgg aac cgg 8392Gly Ser Ala Ala Arg Ser Ile Trp His Gly Phe Val Arg Trp Asn Arg160 165 170ggc gag gcc gag gat ggg cat gac agc cac ggc gtc ccg ctg gac ctg 8440Gly Glu Ala Glu Asp Gly His Asp Ser His Gly Val Pro Leu Asp Leu175 180 185cgc tgg ccc ggc ttc cgc atc gcg atc gtg gcc gtg gac aag ggg ccc 8488Arg Trp Pro Gly Phe Arg Ile Ala Ile Val Ala Val Asp Lys Gly Pro190 195 200aag cct ttc agt tcg cgc gac ggc atg aac cac acg gtc gag acc agc 8536
Lys Pro Phe Ser Ser Arg Asp Gly Met Asn His Thr Val Glu Thr Ser205 210 215ccg ctg ttc ccg ccc tgg cct gcg cag gcg gaa gcg gat tgc cgc gtc 8584Pro Leu Phe Pro Pro Trp Pro Ala Gln Ala Glu Ala Asp Cys Arg Val220 225 230 235atc gag gat gcg atc gcc gcc cgc gac atg gcc gcc ctg ggt ccg cgg 8632Ile Glu Asp Ala Ile Ala Ala Arg Asp Met Ala Ala Leu Gly Pro Arg240 245 250gtc gag gcg aac gcc ctt gcg atg cac gcc acg atg atg gcc gcg cgc 8680Val Glu Ala Asn Ala Leu Ala Met His Ala Thr Met Met Ala Ala Arg255 260 265ccg ccg ctc tgc tac ctg acg ggc ggc agc tgg cag gtg ctg gaa cgc 8728Pro Pro Leu Cys Tyr Leu Thr Gly Gly Ser Trp Gln Val Leu Glu Arg270 275 280ctg tgg cag gcc cgc gcg gac ggg ctt gcg gcc ttt gcg acg atg gat 8776Leu Trp Gln Ala Arg Ala Asp Gly Leu Ala Ala Phe Ala Thr Met Asp285 290 295gcc ggc ccg aac gtc aag ctg atc ttc gag gaa agc agc gcc gcc gac 8824Ala Gly Pro Asn Val Lys Leu Ile Phe Glu Glu Ser Ser Ala Ala Asp300 305 310 315gtg ctg tac ctg ttc ccc gac gcc agc ctg atc gcg ccg ttc gag ggg 8872Val Leu Tyr Leu Phe Pro Asp Ala Ser Leu Ile Ala Pro Phe Glu Gly320 325 330cgt tga acgcgtaaga cgaccactgg gtaaggttct gccgcgcgtg gtctcgactg 8928Argcctgcaaaga ggtgcttgag ttgctgcgtg actgcggcgg ccgacttcgt gggacttgcc8988cgccacgctg acgcgctgga aacgcgcccg cggattacga ccgcgtcatt gccctgaacc9048aatttcccgt cggtcgac 9066<210>53<211>332<212>PRT<213>副球菌属菌种(Paracoccus sp.)<400>53Met Thr Asp Ala Val Arg Asp Met Ile Ala Arg Ala Met Ala Gly Ala1 5 10 15Thr Asp Ile Arg Ala Ala Glu Ala Tyr Ala Pro Ser Asn Ile Ala Leu20 25 30Ser Lys Tyr Trp Gly Lys Arg Asp Ala Ala Arg Asn Leu Pro Leu Asn35 40 45Ser Ser Val Ser Ile Ser Leu Ala Asn Trp Gly Ser His Thr Arg Val50 55 60Glu Gly Ser Gly Thr Gly His Asp Glu Val His His Asn Gly Thr Leu65 70 75 80Leu Asp Pro Gly Asp Ala Phe Ala Arg Arg Ala Leu Ala Phe Ala Asp85 90 95
Leu Phe Arg Gly Gly Arg His Leu Pro Leu Arg Ile Thr Thr Gln Asn100 105 110Ser Ile Pro Thr Ala Ala Gly Leu Ala Ser Ser Ala Ser Gly Phe Ala115 120 125Ala Leu Thr Arg Ala Leu Ala Gly Ala Phe Gly Leu Asp Leu Asp Asp130 135 140Thr Asp Leu Ser Arg Ile Ala Arg Ile Gly Ser Gly Ser Ala Ala Arg145 150 155 160Ser Ile Trp His Gly Phe Val Arg Trp Asn Arg Gly Glu Ala Glu Asp165 170 175Gly His Asp Ser His Gly Val Pro Leu Asp Leu Arg Trp Pro Gly Phe180 185 190Arg Ile Ala Ile Val Ala Val Asp Lys Gly Pro Lys Pro Phe Ser Ser195 200 205Arg Asp Gly Met Asn His Thr Val Glu Thr Ser Pro Leu Phe Pro Pro210 215 220Trp Pro Ala Gln Ala Glu Ala Asp Cys Arg Val Ile Glu Asp Ala Ile225 230 235 240Ala Ala Arg Asp Met Ala Ala Leu Gly Pro Arg Val Glu Ala Asn Ala245 250 255Leu Ala Met His Ala Thr Met Met Ala Ala Arg Pro Pro Leu Cys Tyr260 265 270Leu Thr Gly Gly Ser Trp Gln Val Leu Glu Arg Leu Trp Gln Ala Arg275 280 285Ala Asp Gly Leu Ala Ala Phe Ala Thr Met Asp Ala Gly Pro Asn Val290 295 300Lys Leu Ile Phe Glu Glu Ser Ser Ala Ala Asp Val Leu Tyr Leu Phe305 310 315 320Pro Asp Ala Ser Leu Ile Ala Pro Phe Glu Gly Arg325 330<210>54<211>353<212>PRT<213>链霉菌属菌种(Streptomyces sp.)<400>54Met Thr Glu Thr His Ala Ile Ala Gly Val Pro Met Arg Trp Val Gly1 5 10 15Pro Leu Arg Ile Ser Gly Asn Val Ala Glu Thr Glu Thr Gln Val Pro20 25 30Leu Ala Thr Tyr Glu Ser Pro Leu Trp Pro Ser Val Gly Arg Gly Ala35 40 45Lys Val Ser Arg Leu Thr Glu Lys Gly Ile Val Ala Thr Leu Val Asp
50 55 60Glu Arg Met Thr Arg Ser Val Ile Val Glu Ala Thr Asp Ala Gln Thr65 70 75 80Ala Tyr Met Ala Ala Gln Thr Ile His Ala Arg Ile Asp Glu Leu Arg85 90 95Glu Val Val Arg Gly Cys Ser Arg Phe Ala Gln Leu Ile Asn Ile Lys100 105 110His Glu Ile Asn Ala Asn Leu Leu Phe Ile Arg Phe Glu Phe Thr Thr115 120 125Gly Asp Ala Ser Gly His Asn Met Ala Thr Leu Ala Ser Asp Val Leu130 135 140Leu Gly His Leu Leu Glu Thr Ile Pro Gly Ile Ser Tyr Gly Ser Ile145 150 155 160Ser Gly Asn Tyr Cys Thr Asp Lys Lys Ala Thr Ala Ile Asn Gly Ile165 170 175Leu Gly Arg Gly Lys Asn Val Ile Thr Glu Leu Leu Val Pro Arg Asp180 185 190Val Val Glu Asn Asn Leu His Thr Thr Ala Ala Lys Ile Val Glu Leu195 200 205Asn Ile Arg Lys Asn Leu Leu Gly Thr Leu Leu Ala Gly Gly Ile Arg210 215 220Ser Ala Asn Ala His Phe Ala Asn Met Leu Leu Gly Phe Tyr Leu Ala225 230 235 240Thr Gly Gln Asp Ala Ala Asn Ile Val Glu Gly Ser Gln Gly Val Val245 250 255Met Ala Glu Asp Arg Asp Gly Asp Leu Tyr Phe Ala Cys Thr Leu Pro260 265 270Asn Leu Ile Val Gly Thr Val Gly Asn Gly Lys Gly Leu Gly Phe Val275 280 285Glu Thr Asn Leu Ala Arg Leu Gly Cys Arg Ala Asp Arg Glu Pro Gly290 295 300Glu Asn Ala Arg Arg Leu Ala Val Ile Ala Ala Ala Thr Val Leu Cys305 310 315 320Gly Glu Leu Ser Leu Leu Ala Ala Gln Thr Asn Pro Gly Glu Leu Met325 330 335Arg Ala His Val Gln Leu Glu Arg Asp Asn Lys Thr Ala Lys Val Gly340 345 350Ala
<210>55<211>353<212>PRT<213>灰孢链霉菌(Streptomyces griseosporeus)<400>55Met Thr Glu Ala His Ala Thr Ala Gly Val Pro Met Arg Trp Val Gly1 5 10 15Pro Val Arg Ile Ser Gly Asn Val Ala Thr Ile Glu Thr Gln Val Pro20 25 30Leu Ala Thr Tyr Glu Ser Pro Leu Trp Pro Ser Val Gly Arg Gly Ala35 40 45Lys Val Ser Arg Leu Thr Glu Lys Gly Ile Val Ala Thr Leu Val Asp50 55 60Glu Arg Met Thr Arg Ser Val Leu Val Glu Ala Thr Asp Ala Leu Thr65 70 75 80Ala Leu Ser Ala Ala Arg Thr Ile Glu Ala Arg Ile Asp Glu Leu Arg85 90 95Glu Leu Val Arg Gly Cys Ser Arg Phe Ala Gln Leu Ile Gly Ile Arg100 105 110His Glu Ile Thr Gly Asn Leu Leu Phe Val Arg Phe Glu Phe Ser Thr115 120 125Gly Asp Ala Ser Gly His Asn Met Ala Thr Leu Ala Ser Asp Val Leu130 135 140Leu Gln His Leu Leu Glu Thr Val Pro Gly Ile Ser Tyr Gly Ser Ile145 150 155 160Ser Gly Asn Tyr Cys Thr Asp Lys Lys Ala Thr Ala Ile Asn Gly Ile165 170 175Leu Gly Arg Gly Lys Asn Val Val Thr Glu Leu Leu Val Pro Arg Asp180 185 190Val Val Ala Asp Val Leu Asn Thr Thr Ala Ala Lys Ile Ala Glu Leu195 200 205Asn Leu Arg Lys Asn Leu Leu Gly Thr Leu Leu Ala Gly Gly Ile Arg210 215 220Ser Ala Asn Ala His Tyr Ala Asn Met Leu Leu Ala Phe Tyr Leu Ala225 230 235 240Thr Gly Gln Asp Ala Ala Asn Ile Val Glu Gly Ser Gln Gly Val Val245 250 255Thr Ala Glu Asp Arg Asp Gly Asp Leu Tyr Leu Ala Cys Thr Leu Pro260 265 270Asn Leu Ile Val Gly Thr Val Gly Asn Gly Lys Gly Leu Gly Phe Val275 280 285Glu Thr Asn Leu Asn Arg Leu Gly Cys Arg Ala Asp Arg Glu Pro Gly290 295 300
Glu Asn Ala Arg Arg Leu Ala Val Ile Ala Ala Ala Thr Val Leu Cys305 310 315 320Gly Glu Leu Ser Leu Leu Ala Ala Gln Thr Asn Pro Gly Glu Leu Met325 330 335Arg Ala His Val Gln Leu Glu Arg Gly His Thr Thr Ala Lys Ala Gly340 345 350Val<210>56<211>353<212>PRT<213>链霉菌属菌种(Streptomyces sp.)<400>56Met Thr Asp Thr His Ala Ile Ala Met Val Pro Met Lys Trp Val Gly1 5 10 15Pro Leu Arg Ile Ser Gly Asn Val Ala Thr Thr Glu Thr His Val Pro20 25 30Leu Ala Thr Tyr Glu Thr Pro Leu Trp Pro Ser Val Gly Arg Gly Ala35 40 45Lys Val Ser Met Leu Ser Glu Arg Gly Ile Ala Ala Thr Leu Val Asp50 55 60Glu Arg Met Thr Arg Ser Val Leu Val Glu Ala Thr Asp Ala Gln Thr65 70 75 80Ala Tyr Thr Ala Ala Arg Ala Ile Glu Ala Arg Ile Glu Glu Leu Arg85 90 95Ala Val Val Arg Thr Cys Ser Arg Phe Ala Glu Leu Leu Gln Val Arg100 105 110His Glu Ile Ala Gly Asn Leu Leu Phe Val Arg Phe Glu Phe Ser Thr115 120 125Arg Arg Pro Ser Gly His Asn Met Ala Thr Leu Ala Ser Asp Ala Leu130 135 140Leu Ala His Leu Leu Gln Thr Ile Pro Gly Ile Ser Tyr Gly Ser Ile145 150 155 160Ser Gly Asn Tyr Cys Thr Asp Lys Lys Ala Thr Ala Ile Asn Gly Ile165 170 175Leu Gly Arg Gly Lys Asn Val Val Thr Glu Leu Val Val Pro Arg Glu180 185 190Val Val Glu Arg Val Leu His Thr Thr Ala Ala Lys Ile Val Glu Leu195 200 205Asn Ile Arg Lys Asn Leu Leu Gly Thr Leu Leu Ala Gly Gly Ile Arg210 215 220Ser Ala Asn Ala His Tyr Ala Asn Met Leu Leu Gly Phe Tyr Leu Ala
225 230 235 240Thr Gly Gln Asp Ala Ala Asn Ile Val Glu Gly Ser Gln Gly Val Thr245 250 255Leu Ala Glu Asp Arg Asp Gly Asp Leu Tyr Phe Ser Cys Asn Leu Pro260 265 270Asn Leu Ile Val Gly Thr Val Gly Asn Gly Lys Gly Leu Glu Phe Val275 280 285Glu Thr Asn Leu Asn Arg Leu Gly Cys Arg Glu Asp Arg Ala Pro Gly290 295 300Glu Asn Ala Arg Arg Leu Ala Val Ile Ala Ala Ala Thr Val Leu Cys305 310 315 320Gly Glu Leu Ser Leu Leu Ala Ala Gln Thr Asn Pro Gly Glu Leu Met325 330 335Arg Ala His Val Glu Leu Glu Arg Asp Asn Thr Thr Ala Glu Val Gly340 345 350Val<210>57<211>347<212>PRT<213>草生欧文氏菌(Erwinia herbicola)<400>57Met Lys Asp Glu Arg Leu Val Gln Arg Lys Asn Asp His Leu Asp Ile1 5 10 15Val Leu Asp Pro Arg Arg Ala Val Thr Gln Ala Ser Ala Gly Phe Glu20 25 30Arg Trp Arg Phe Thr His Cys Ala Leu Pro Glu Leu Asn Phe Ser Asp35 40 45Ile Thr Leu Glu Thr Thr Phe Leu Asn Arg Gln Leu Gln Ala Pro Leu50 55 60Leu Ile Ser Ser Met Thr Gly Gly Val Glu Arg Ser Arg His Ile Asn65 70 75 80Arg His Leu Ala Glu Ala Ala Gln Val Leu Lys Ile Ala Met Gly Val85 90 95Gly Ser Gln Arg Val Ala Ile Glu Ser Asp Ala Gly Leu Gly Leu Asp100 105 110Lys Thr Leu Arg Gln Leu Ala Pro Asp Val Pro Leu Leu Ala Asn Leu115 120 125Gly Ala Ala Gln Leu Thr Gly Arg Lys Gly Ile Asp Tyr Ala Arg Arg130 135 140Ala Val Glu Met Ile Glu Ala Asp Ala Leu Ile Val His Leu Asn Pro145 150 155 160
Leu Gln Glu Ala Leu Gln Pro Gly Gly Asp Arg Asp Trp Arg Gly Arg165 170 175Leu Ala Ala Ile Glu Thr Leu Val Arg Glu Leu Pro Val Pro Leu Val180 185 190Val Lys Glu Val Gly Ala Gly Ile Ser Arg Thr Val Ala Gly Gln Leu195 200 205Ile Asp Ala Gly Val Thr Val Ile Asp Val Ala Gly Ala Gly Gly Thr210 215 220Ser Trp Ala Ala Val Glu Gly Glu Arg Ala Ala Thr Glu Gln Gln Arg225 230 235 240Ser Val Ala Asn Val Phe Ala Asp Trp Gly Ile Pro Thr Ala Glu Ala245 250 255Leu Val Asp Ile Ala Glu Ala Trp Pro Gln Met Pro Leu Ile Ala Ser260 265 270Gly Gly Ile Lys Asn Gly Val Asp Ala Ala Lys Ala Leu Arg Leu Gly275 280 285Ala Cys Met Val Gly Gln Ala Ala Ala Val Leu Gly Ser Ala Gly Val290 295 300Ser Thr Glu Lys Val Ile Asp His Phe Asn Val Ile Ile Glu Gln Leu305 310 315 320Arg Val Ala Cys Phe Cys Thr Gly Ser Arg Ser Leu Ser Asp Leu Lys325 330 335Gln Ala Asp Ile Arg Tyr Val Arg Asp Thr Pro340 345<210>58<211>360<212>PRT<213>布氏疏螺旋体(Borrelia burgdorferi)<400>58Met Met Asp Thr Glu Phe Met Gly Ile Glu Pro Asn Ile Leu Glu Asn1 5 10 15Lys Lys Arg His Ile Glu Ile Cys Leu Asn Lys Asn Asp Val Lys Gly20 25 30Gly Cys Asn Phe Leu Lys Phe Ile Lys Leu Lys His Asn Ala Leu Ser35 40 45Asp Phe Asn Phe Ser Glu Ile Asn Ile Lys Glu Glu Ile Phe Gly Tyr50 55 60Asn Ile Ser Met Pro Val Phe Ile Ser Ser Met Thr Gly Gly Ser Lys65 70 75 80Glu Gly Asn Asp Phe Asn Lys Ser Leu Val Arg Ile Ala Asn Tyr Leu85 90 95
Lys Ile Pro Ile Gly Leu Gly Ser Phe Lys Leu Leu Phe Lys Tyr Pro100 105 110Glu Tyr Ile Arg Asp Phe Thr Leu Lys Arg Tyr Ala His Asn Ile Pro115 120 125Leu Phe Ala Asn Val Gly Ala Val Gln Ile Val Glu Phe Gly Ile Ser130 135 140Lys Ile Ala Glu Met Ile Lys Arg Leu Glu Val Asp Ala Ile Ile Val145 150 155 160His Leu Asn Ala Gly Gln Glu Leu Met Lys Val Asp Gly Asp Arg Asn165 170 175Phe Lys Gly Ile Arg Glu Ser Ile Ala Lys Leu Ser Asp Phe Leu Ser180 185 190Val Pro Leu Ile Val Lys Glu Thr Gly Phe Gly Ile Ser Pro Lys Asp195 200 205Val Lys Glu Leu Phe Ser Leu Gly Ala Ser Tyr Val Asp Leu Ala Gly210 215 220Ser Gly Gly Thr Asn Trp Ile Leu Val Glu Gly Met Lys Ser Asn Asn225 230 235 240Leu Asn Ile Ala Ser Cys Phe Ser Asp Trp Gly Ile Pro Ser Val Phe245 250 255Thr Leu Leu Ser Ile Asp Asp Ser Leu Lys Ala Asn Ile Phe Ala Ser260 265 270Gly Gly Tyr Glu Thr Gly Met Asp Ile Ala Lys Gly Ile Ala Leu Gly275 280 285Ala Arg Leu Ile Gly Val Ala Ala Val Val Leu Arg Ala Phe Tyr Asp290 295 300Ser Gly Glu Asp Ala Val Phe Gly Leu Phe Ser Asp Tyr Glu His Ile305 310 315 320Leu Lys Met Ser Met Phe Leu Ser Gly Ser Lys Ser Leu Leu Glu Phe325 330 335Arg Asn Asn Lys Tyr Phe Leu Ser Ser Tyr Leu Leu Asp Glu Leu Gly340 345 350Val Phe Lys Gln Phe Tyr Gly Thr355 360<210>59<211>349<212>PRT<213>集胞蓝细菌属菌种(Synechocystis sp.)<400>59Met Asp Ser Thr Pro His Arg Lys Ser Asp His Ile Arg Ile Val Leu1 5 10 15Glu Glu Asp Val Val Gly Lys Gly Ile Ser Thr Gly Phe Glu Arg Leu
20 25 30Met Leu Glu His Cys Ala Leu Pro Ala Val Asp Leu Asp Ala Val Asp35 40 45Leu Gly Leu Thr Leu Trp Gly Lys Ser Leu Thr Tyr Pro Trp Leu Ile50 55 60Ser Ser Met Thr Gly Gly Thr Pro Glu Ala Lys Gln Ile Asn Leu Phe65 70 75 80Leu Ala Glu Val Ala Gln Ala Leu Gly Ile Ala Met Gly Leu Gly Ser85 90 95Gln Arg Ala Ala Ile Glu Asn Pro Asp Leu Ala Phe Thr Tyr Gln Val100 105 110Arg Ser Val Ala Pro Asp Ile Leu Leu Phe Ala Asn Leu Gly Leu Val115 120 125Gln Leu Asn Tyr Gly Tyr Gly Leu Glu Gln Ala Gln Arg Ala Val Asp130 135 140Met Ile Glu Ala Asp Ala Leu Ile Leu His Leu Asn Pro Leu Gln Glu145 150 155 160Ala Val Gln Pro Asp Gly Asp Arg Leu Trp Ser Gly Leu Trp Ser Lys165 170 175Leu Glu Ala Leu Val Glu Ala Leu Glu Val Pro Val Ile Val Lys Glu180 185 190Val Gly Asn Gly Ile Ser Gly Pro Val Ala Lys Arg Leu Gln Glu Cys195 200 205Gly Val Gly Ala Ile Asp Val Ala Gly Ala Gly Gly Thr Ser Trp Ser210 215 220Glu Val Glu Ala His Arg Gln Thr Asp Arg Gln Ala Lys Glu Val Ala225 230 235 240His Asn Phe Ala Asp Trp Gly Leu Pro Thr Ala Trp Ser Leu Gln Gln245 250 255Val Val Gln Asn Thr Glu Gln Ile Leu Val Phe Ala Ser Gly Gly Ile260 265 270Arg Ser Gly Ile Asp Gly Ala Lys Ala Ile Ala Leu Gly Ala Thr Leu275 280 285Val Gly Ser Ala Ala Pro Val Leu Ala Glu Ala Lys Ile Asn Ala Gln290 295 300Arg Val Tyr Asp His Tyr Gln Ala Arg Leu Arg Glu Leu Gln Ile Ala305 310 315 320Ala Phe Cys Cys Asp Ala Ala Asn Leu Thr Gln Leu Ala Gln Val Pro325 330 335Leu Trp Asp Arg Gln Ser Gly Gln Arg Leu Thr Lys Pro340 345
<210>60<211>361<212>PRT<213>链霉菌属菌种(Streptomyces sp.)<400>60Met Thr Ser Ala Gln Arg Lys Asp Asp His Val Arg Leu Ala Ile Glu1 5 10 15Gln His Asn Ala His Ser Gly Arg Asn Gln Asp Asp Val Ser Phe Val20 25 30His His Ala Leu Ala Gly Ile Asp Arg Pro Asp Val Ser Leu Ala Thr35 40 45Ser Phe Ala Gly Ile Ser Trp Gln Val Pro Ile Tyr Ile Asn Ala Met50 55 60Thr Gly Gly Ser Glu Lys Thr Gly Leu Ile Asn Arg Asp Leu Ala Thr65 70 75 80Ala Ala Arg Glu Thr Gly Val Pro Ile Ala Ser Gly Ser Met Asn Ala85 90 95Tyr Ile Lys Asp Pro Cys Ala Asp Thr Phe Arg Val Leu Arg Asp Glu100 105 110Asn Pro Asn Gly Phe Val Ile Ala Asn Ile Asn Ala Thr Thr Thr Val115 120 125Asp Asn Ala Gln Arg Ala Ile Asp Leu Ile Glu Ala Asn Ala Leu Gln130 135 140Ile His Ile Asn Thr Ala Gln Glu Thr Pro Met Pro Glu Gly Asp Arg145 150 155 160Ser Phe Ala Ser Trp Val Pro Gln Ile Glu Lys Ile Ala Ala Ala Val165 170 175Asp Ile Pro Val Ile Val Lys Glu Val Gly Asn Gly Leu Ser Arg Gln180 185 190Thr Ile Leu Leu Leu Ala Asp Leu Gly Val Gln Ala Ala Asp Val Ser195 200 205Gly Arg Gly Gly Thr Asp Phe Ala Arg Ile Glu Asn Gly Arg Arg Glu210 215 220Leu Gly Asp Tyr Ala Phe Leu His Gly Trp Gly Gln Ser Thr Ala Ala225 230 235 240Cys Leu Leu Asp Ala Gln Asp Ile Ser Leu Pro Val Leu Ala Ser Gly245 250 255Gly Val Arg His Pro Leu Asp Val Val Arg Ala Leu Ala Leu Gly Ala260 265 270Arg Ala Val Gly Ser Ser Ala Gly Phe Leu Arg Thr Leu Met Asp Asp275 280 285
Gly Val Asp Ala Leu Ile Thr Lys Leu Thr Thr Trp Leu Asp Gln Leu290 295 300Ala Ala Leu Gln Thr Met Leu Gly Ala Arg Thr Pro Ala Asp Leu Thr305 310 315 320Arg Cys Asp Val Leu Leu His Gly Glu Leu Arg Asp Phe Cys Ala Asp325 330 335Arg Gly Ile Asp Thr Arg Arg Leu Ala Gln Arg Ser Ser Ser Ile Glu340 345 350Ala Leu Gln Thr Thr Gly Ser Thr Arg355 360<210>61<211>364<212>PRT<213>灰孢链霉菌(Streptomyces griseosporeus)<400>61Met Ser Ser Ala Gln Arg Lys Asp Asp His Val Arg Leu Ala Thr Glu1 5 10 15Gln Gln Arg Ala His Ser Gly Arg Asn Gln Phe Asp Asp Val Ser Phe20 25 30Val His His Ala Leu Ala Gly Ile Asp Arg Pro Asp Val Arg Leu Ala35 40 45Thr Thr Phe Ala Gly Ile Thr Trp Arg Leu Pro Leu Tyr Ile Asn Ala50 55 60Met Thr Gly Gly Ser Ala Lys Thr Gly Ala Ile Asn Arg Asp Leu Ala65 70 75 80Val Ala Ala Arg Glu Thr Gly Ala Ala Ile Ala Ser Gly Ser Met His85 90 95Ala Phe Phe Arg Asp Pro Ser Cys Ala Asp Thr Phe Arg Val Leu Arg100 105 110Thr Glu Asn Pro Asp Gly Phe Val Met Ala Asn Val Asn Ala Thr Ala115 120 125Ser Val Asp Asn Ala Arg Arg Ala Val Asp Leu Ile Glu Ala Asn Ala130 135 140Leu Gln Ile His Leu Asn Thr Ala Gln Glu Thr Pro Met Pro Glu Gly145 150 155 160Asp Arg Ser Phe Gly Ser Trp Pro Ala Gln Ile Ala Lys Ile Thr Ala165 170 175Ala Val Asp Val Pro Val Ile Val Lys Glu Val Gly Asn Gly Leu Ser180 185 190Arg Gln Thr Leu Leu Ala Leu Pro Asp Leu Gly Val Arg Val Ala Asp195 200 205Val Ser Gly Arg Gly Gly Thr Asp Phe Ala Arg Ile Glu Asn Ser Arg210 215 220
Arg Pro Leu Gly Asp Tyr Ala Phe Leu His Gly Trp Gly Gln Ser Thr225 230 235 240Pro Ala Cys Leu Leu Asp Ala Gln Asp Val Gly Phe Pro Leu Leu Ala245 250 255Ser Gly Gly Ile Arg Asn Pro Leu Asp Val Ala Arg Ala Leu Ala Leu260 265 270Gly Ala Gly Ala Val Gly Ser Ser Gly Val Phe Leu Arg Thr Leu Ile275 280 285Asp Gly Gly Val Ser Ala Leu Val Ala Gln Ile Ser Thr Trp Leu Asp290 295 300Gln Leu Ala Ala Leu Gln Thr Met Leu Gly Ala Arg Thr Pro Ala Asp305 310 315 320Leu Thr Arg Cys Asp Val Leu Ile His Gly Pro Leu Arg Ser Phe Cys325 330 335Thr Asp Arg Gly Ile Asp Ile Gly Arg Phe Ala Arg Arg Ser Ser Ser340 345 350Ala Asp Ile Arg Ser Glu Met Thr Gly Ser Thr Arg355 360<210>62<211>368<212>PRT<213>硫磺矿硫化叶菌(Sulfolobus solfataricus)<400>62Met Pro Asp Ile Val Asn Arg Lys Val Glu His Val Glu Ile Ala Ala1 5 10 15Phe Glu Asn Val Asp Gly Leu Ser Ser Ser Thr Phe Leu Asn Asp Val20 25 30Ile Leu Val His Gln Gly Phe Pro Gly Ile Ser Phe Ser Glu Ile Asn35 40 45Thr Lys Thr Lys Phe Phe Arg Lys Glu Ile Ser Ala Pro Ile Met Val50 55 60Thr Gly Met Thr Gly Gly Arg Asn Glu Leu Gly Arg Ile Asn Arg Ile65 70 75 80Ile Ala Glu Val Ala Glu Lys Phe Gly Ile Pro Met Gly Val Gly Ser85 90 95Gln Arg Val Ala Ile Glu Lys Ala Glu Ala Arg Glu Ser Phe Thr Ile100 105 110Val Arg Lys Val Ala Pro Thr Ile Pro Ile Ile Ala Asn Leu Gly Met115 120 125Pro Gln Leu Val Lys Gly Tyr Gly Leu Lys Glu Phe Gln Asp Ala Ile130 135 140
Gln Met Ile Glu Ala Asp Ala Ile Ala Val His Leu Asn Pro Ala Gln145 150 155 160Glu Val Phe Gln Pro Glu Gly Glu Pro Glu Tyr Gln Ile Tyr Ala Leu165 170 175Glu Arg Leu Arg Asp Ile Ser Lys Glu Leu Ser Val Pro Ile Ile Val180 185 190Lys Glu Ser Gly Asn Gly Ile Ser Met Glu Thr Ala Lys Leu Leu Tyr195 200 205Ser Tyr Gly Ile Lys Asn Phe Asp Thr Ser Gly Gln Gly Gly Thr Asn210 215 220Trp Ile Ala Ile Glu Met Ile Arg Asp Ile Arg Arg Gly Asn Trp Lys225 230 235 240Ala Glu Ser Ala Lys Asn Phe Leu Asp Trp Gly Val Pro Thr Ala Ala245 250 255Ser Ile Ile Glu Val Arg Tyr Ser Ile Pro Asp Ala Phe Leu Val Gly260 265 270Ser Gly Gly Ile Arg Ser Gly Leu Asp Ala Ala Lys Ala Ile Ala Leu275 280 285Gly Ala Asp Ile Ala Gly Met Ala Leu Pro Val Leu Lys Ser Ala Ile290 295 300Glu Gly Lys Glu Ser Leu Glu Gln Phe Phe Arg Lys Ile Ile Phe Glu305 310 315 320Leu Lys Ala Thr Met Met Leu Thr Gly Ser Lys Asn Val Glu Ala Leu325 330 335Lys Arg Ser Ser Ile Val Ile Leu Gly Lys Leu Lys Glu Trp Ala Glu340 345 350Tyr Arg Gly Ile Asn Leu Ser Ile Tyr Glu Lys Val Arg Lys Arg Glu355 360 365<210>63<211>342<212>PRT<213>普氏立克次氏体(Rickettsia prowazekii)<400>63Met Pro Lys Glu Gln Asn Leu Asp Ile Glu Arg Lys Gln Glu His Ile1 5 10 15Glu Ile Asn Leu Lys Gln Asn Val Asn Ser Thr Leu Lys Ser Gly Leu20 25 30Glu Ser Ile Lys Phe Ile His Asn Ala Leu Pro Glu Ile Asn Tyr Asp35 40 45Ser Ile Asp Thr Thr Thr Thr Phe Leu Gly Lys Asp Met Lys Ala Pro50 55 60Ile Leu Ile Ser Ser Met Thr Gly Gly Thr Ala Arg Ala Arg Asp Ile65 70 75 80
Asn Tyr Arg Leu Ala Gln Ala Ala Gln Lys Ser Gly Ile Ala Met Gly85 90 95Leu Gly Ser Met Arg Ile Leu Leu Thr Lys Pro Asp Thr Ile Lys Thr100 105 110Phe Thr Val Arg His Val Ala Pro Asp Ile Pro Leu Leu Ala Asn Ile115 120 125Gly Ala Val Gln Leu Asn Tyr Gly Val Thr Pro Lys Glu Cys Gln Tyr130 135 140Leu Ile Asp Thr Ile Lys Ala Asp Ala Leu Ile Leu His Leu Asn Val145 150 155 160Leu His Glu Leu Thr Gln Pro Glu Gly Asn Lys Asn Trp Glu Asn Leu165 170 175Leu Pro Lys Ile Lys Glu Val Ile Asn Tyr Leu Ser Val Pro Val Ile180 185 190Val Lys Glu Val Gly Tyr Gly Leu Ser Lys Gln Val Ala Lys Lys Leu195 200 205Ile Lys Ala Gly Val Lys Val Leu Asp Ile Ala Gly Ser Gly Gly Thr210 215 220Ser Trp Ser Gln Val Glu Ala Tyr Arg Ala Lys Asn Ser Met Gln Asn225 230 235 240Arg Ile Ala Ser Ser Phe Ile Asn Trp Gly Ile Thr Thr Leu Asp Ser245 250 255Leu Lys Met Leu Gln Glu Ile Ser Lys Asp Ile Thr Ile Ile Ala Ser260 265 270Gly Gly Leu Gln Ser Gly Ile Asp Gly Ala Lys Ala Ile Arg Met Gly275 280 285Ala Asn Ile Phe Gly Leu Ala Gly Lys Leu Leu Lys Ala Ala Asp Ile290 295 300Ala Glu Ser Leu Val Leu Glu Glu Ile Gln Val Ile Ile Glu Gln Leu305 310 315 320Lys Ile Thr Met Leu Cys Thr Gly Ser Cys Thr Leu Lys Asp Leu Ala325 330 335Lys Ala Glu Ile Met Trp340<210>64<211>286<212>PRT<213>抗放射异常球菌(Deinococcus radiodurans)<400>64Met Arg Leu Asp Thr Val Phe Leu Gly Arg Arg Leu Lys Ala Pro Val1 5 10 15
Leu Ile Gly Ala Met Thr Gly Gly Ala Glu Lys Ala Gly Val Ile Asn20 25 30Arg Asn Leu Ala Thr Ala Ala Arg Asn Leu Gly Leu Gly Met Met Leu35 40 45Gly Ser Gln Arg Val Met Leu Glu His Pro Asp Ala Trp Glu Ser Phe50 55 60Asn Val Arg Glu Val Ala Pro Glu Ile Leu Leu Ile Gly Asn Leu Gly65 70 75 80Ala Ala Gln Phe Met Leu Gly Tyr Gly Ala Glu Gln Ala Arg Arg Ala85 90 95Val Asp Glu Val Met Ala Asp Ala Leu Ala Ile His Leu Asn Pro Leu100 105 110Gln Glu Ala Leu Gln Arg Gly Gly Asp Thr Arg Trp Gln Gly Val Thr115 120 125Tyr Arg Leu Lys Gln Val Ala Arg Glu Leu Asp Phe Pro Val Ile Ile130 135 140Lys Glu Val Gly His Gly Leu Asp Ala Ala Thr Leu Arg Ala Leu Ala145 150 155 160Asp Gly Pro Phe Ala Ala Tyr Asp Val Ala Gly Ala Gly Gly Thr Ser165 170 175Trp Ala Arg Val Glu Gln Leu Val Ala His Gly Gln Val His Ser Pro180 185 190Asp Leu Cys Glu Leu Gly Val Pro Thr Ala Gln Ala Leu Arg Gln Ala195 200 205Arg Lys Thr Leu Pro Gly Ala Gln Leu Ile Ala Ser Gly Gly Ile Arg210 215 220Ser Gly Leu Asp Ala Ala Arg Ala Leu Ser Leu Gly Ala Glu Val Val225 230 235 240Ala Val Ala Arg Pro Leu Leu Glu Pro Ala Leu Asp Ser Ser Glu Ala245 250 255Ala Glu Ala Trp Leu Arg Asn Phe Ile Gln Glu Leu Arg Val Ala Leu260 265 270Phe Val Gly Gly Tyr Arg Asp Val Arg Glu Val Arg Gly Gly275 280 285<210>65<211>361<212>PRT<213>Aeropyrum pernix<400>65Met Ile Val Ser Ser Lys Val Glu Ser Arg Glu Ser Thr Leu Leu Glu1 5 10 15Tyr Val Arg Ile Val His Asn Pro Thr Pro Glu Val Asn Leu Gly Asp20 25 30
Val Ser Leu Glu Ile Asp Phe Cys Gly Gly Arg Leu Arg Ala Pro Leu35 40 45Val Ile Thr Gly Met Thr Gly Gly His Pro Asp Val Glu Trp Ile Asn50 55 60Arg Glu Leu Ala Ser Val Ala Glu Glu Leu Gly Ile Ala Ile Gly Val65 70 75 80Gly Ser Gln Arg Ala Ala Ile Glu Asp Pro Ser Leu Ala Arg Thr Phe85 90 95Arg Ala Ala Arg Glu Ala Ala Pro Asn Ala Phe Leu Ile Ala Asn Leu100 105 110Gly Ala Pro Gln Leu Ser Leu Gly Tyr Ser Val Arg Glu Val Arg Met115 120 125Ala Val Glu Met Ile Asp Ala Asp Ala Ile Ala Ile His Leu Asn Pro130 135 140Gly Gln Glu Ala Tyr Gln Pro Glu Gly Asp Pro Phe Tyr Arg Gly Val145 150 155 160Val Gly Lys Ile Ala Glu Ala Ala Glu Ala Ala Gly Val Pro Val Ile165 170 175Val Lys Glu Thr Gly Asn Gly Leu Ser Arg Glu Ala Val Ala Gln Leu180 185 190Arg Ala Leu Gly Val Arg Cys Phe Asp Val Ala Gly Leu Gly Gly Thr195 200 205Asn Trp Ile Lys Ile Glu Val Leu Arg Gly Arg Lys Ala Gly Ser Pro210 215 220Leu Glu Ala Gly Pro Leu Gln Asp Phe Trp Gly Asn Pro Thr Ala Ala225 230 235 240Ala Leu Met Glu Ala Arg Thr Ala Ala Pro Asp Ala Tyr Ile Ile Ala245 250 255Ser Gly Gly Val Arg Asn Gly Leu Asp Ala Ala Arg Ala Ile Ala Leu260 265 270Gly Ala Asp Ala Ala Gly Val Ala Leu Pro Ala Ile Arg Ser Leu Leu275 280 285Ser Gly Gly Arg Gln Ala Thr Leu Lys Leu Leu Lys Ala Ile Glu Tyr290 295 300Gln Leu Lys Thr Ala Val Tyr Met Val Gly Glu Thr Arg Val Arg Gly305 310 315 320Leu Trp Arg Ala Pro Ile Val Val Trp Gly Arg Leu Ala Glu Glu Ala325 330 335Glu Ala Arg Gly Ile Asp Pro Arg Trp Tyr Thr Asn Thr Leu Arg Leu
340 345 350Glu Ala Leu Val Tyr Lys Asp Val Lys355 360<210>66<211>379<212>PRT<213>盐杆菌属菌种(Halobacterium sp.)<400>66Met Gly Glu Ser Arg Tyr Asn Ser Ile Val Phe Pro Ser Leu Val Gln1 5 10 15Thr Arg Leu Met Thr Ala Gln Asp Ser Thr Gln Thr Glu Asp Arg Lys20 25 30Asp Asp His Leu Gln Ile Val Gln Glu Arg Asp Val Glu Thr Thr Gly35 40 45Thr Gly Phe Asp Asp Val His Leu Val His Asn Ala Leu Pro Glu Leu50 55 60Asp Tyr Asp Ala Ile Asp Pro Ser Ile Asp Phe Leu Gly His Asp Leu65 70 75 80Ser Ala Pro Ile Phe Ile Glu Ser Met Thr Gly Gly His His Asn Thr85 90 95Thr Glu Ile Asn Arg Ala Leu Ala Arg Ala Ala Ser Glu Thr Gly Ile100 105 110Ala Met Gly Leu Gly Ser Gln Arg Ala Gly Leu Glu Leu Asp Asp Glu115 120 125Arg Val Leu Glu Ser Tyr Thr Val Val Arg Asp Ala Ala Pro Asp Ala130 135 140Phe Ile Tyr Gly Asn Leu Gly Ala Ala Gln Leu Arg Glu Tyr Asp Ile145 150 155 160Glu Met Val Glu Gln Ala Val Glu Met Ile Asp Ala Asp Ala Leu Ala165 170 175Val His Leu Asn Phe Leu Gln Glu Ala Thr Gln Pro Glu Gly Asp Val180 185 190Asp Gly Arg Asn Cys Val Ala Ala Ile Glu Arg Val Ser Glu Ala Leu195 200 205Ser Val Pro Ile Ile Val Lys Glu Thr Gly Asn Gly Ile Ser Gly Glu210 215 220Thr Ala Arg Glu Leu Thr Ala Ala Gly Val Asp Ala Leu Asp Val Ala225 230 235 240Gly Lys Gly Gly Thr Thr Trp Ser Gly Ile Glu Ala Tyr Arg Ala Ala245 250 255Ala Ala Asn Ala Pro Arg Gln Lys Gln Ile Gly Thr Leu Phe Arg Glu260 265 270
Trp Gly Ile Pro Thr Ala Ala Ser Thr Ile Glu Cys Val Ala Glu His275 280 285Asp Cys Val Ile Ala Ser Gly Gly Val Arg Thr Gly Leu Asp Val Ala290 295 300Lys Ala Ile Ala Leu Gly Ala Arg Ala Gly Gly Leu Ala Lys Pro Phe305 310 315 320Leu Lys Pro Ala Thr Asp Gly Pro Asp Ala Val Ile Glu Arg Val Gly325 330 335Asp Leu Ile Ala Glu Leu Arg Thr Ala Met Phe Val Thr Gly Ser Gly340 345 350Ser Ile Asp Glu Leu Gln Gln Val Glu Tyr Val Leu His Gly Lys Thr355 360 365Arg Glu Tyr Val Glu Gln Arg Thr Ser Ser Glu370 375<210>67<211>317<212>PRT<213>闪烁古生球菌(Archaeoglobus fulgidus)<400>67Met Met Leu Ile His Lys Ala Leu Pro Glu Val Asp Tyr Trp Lys Ile1 5 10 15Asp Thr Glu Ile Glu Phe Phe Gly Lys Lys Leu Ser Phe Pro Leu Leu20 25 30Ile Ala Ser Met Thr Gly Gly His Pro Glu Thr Lys Glu Ile Asn Ala35 40 45Arg Leu Gly Glu Ala Val Glu Glu Ala Gly Ile Gly Met Gly Val Gly50 55 60Ser Gln Arg Ala Ala Ile Glu Asp Glu Ser Leu Ala Asp Ser Phe Thr65 70 75 80Val Val Arg Glu Lys Ala Pro Asn Ala Phe Val Tyr Ala Asn Ile Gly85 90 95Met Pro Gln Val Ile Glu Arg Gly Val Glu Ile Val Asp Arg Ala Val100 105 110Glu Met Ile Asp Ala Asp Ala Val Ala Ile His Leu Asn Tyr Leu Gln115 120 125Glu Ala Ile Gln Pro Glu Gly Asp Leu Asn Ala Glu Lys Gly Leu Glu130 135 140Val Leu Glu Glu Val Cys Arg Ser Val Lys Val Pro Val Ile Ala Lys145 150 155 160Glu Thr Gly Ala Gly Ile Ser Arg Glu Val Ala Val Met Leu Lys Arg165 170 175Ala Gly Val Ser Ala Ile Asp Val Gly Gly Lys Gly Gly Thr Thr Phe
180 185 190Ser Gly Val Glu Val Tyr Arg Val Asn Asp Glu Val Ser Lys Ser Val195 200 205Gly Ile Asp Phe Trp Asp Trp Gly Leu Pro Thr Ala Phe Ser Ile Val210 215 220Asp Cys Arg Gly Ile Leu Pro Val Ile Ala Thr Gly Gly Leu Arg Ser225 230 235 240Gly Leu Asp Val Ala Lys Ser Ile Ala Ile Gly Ala Glu Leu Gly Ser245 250 255Ala Ala Leu Pro Phe Leu Arg Ala Ala Val Glu Ser Ala Glu Lys Val260 265 270Arg Glu Glu Ile Glu Tyr Phe Arg Arg Gly Leu Lys Thr Ala Met Phe275 280 285Leu Thr Gly Cys Lys Asn Val Glu Glu Leu Lys Gly Leu Lys Val Phe290 295 300Val Ser Gly Arg Leu Lys Glu Trp Ile Asp Phe Arg Gly305 310 315<210>68<211>370<212>PRT<213>Pyrococcus abyssi<400>68Met Glu Glu Gln Thr Ile Leu Arg Lys Phe Glu His Ile Lys His Cys1 5 10 15Leu Thr Lys Asn Val Glu Ala His Val Thr Asn Gly Phe Glu Asp Val20 25 30His Leu Ile His Lys Ser Leu Pro Glu Ile Asp Lys Asp Glu Ile Asp35 40 45Leu Ser Val Lys Phe Leu Gly Arg Lys Phe Asp Tyr Pro Ile Met Ile50 55 60Thr Gly Met Thr Gly Gly Thr Arg Lys Gly Glu Ile Ala Trp Arg Ile65 70 75 80Asn Arg Thr Leu Ala Gln Ala Ala Gln Glu Leu Asn Ile Pro Leu Gly85 90 95Leu Gly Ser Gln Arg Ala Met Ile Glu Lys Pro Glu Thr Trp Glu Ser100 105 110Tyr Tyr Val Arg Asp Val Ala Pro Asp Val Phe Leu Val Gly Asn Leu115 120 125Gly Ala Pro Gln Phe Gly Arg Asn Ala Lys Lys Arg Tyr Ser Val Asp130 135 140Glu Val Leu Tyr Ala Ile Glu Lys Ile Glu Ala Asp Ala Ile Ala Ile145 150 155 160
His Met Asn Pro Leu Gln Glu Ser Ile Gln Pro Glu Gly Asp Thr Thr165 170 175Phe Ser Gly Val Leu Glu Ala Leu Ala Glu Ile Thr Ser Thr Ile Asp180 185 190Tyr Pro Val Ile Ala Lys Glu Thr Gly Ala Gly Val Ser Lys Glu Val195 200 205Ala Val Glu Leu Glu Ala Val Gly Val Asp Ala Ile Asp Ile Ser Gly210 215 220Leu Gly Gly Thr Ser Trp Ser Ala Val Glu Tyr Tyr Arg Thr Lys Asp225 230 235 240Gly Glu Lys Arg Asn Leu Ala Leu Lys Phe Trp Asp Trp Gly Ile Lys245 250 255Thr Ala Ile Ser Leu Ala Glu Val Arg Trp Ala Thr Asn Leu Pro Ile260 265 270Ile Ala Ser Gly Gly Met Arg Asp Gly Ile Thr Met Ala Lys Ala Leu275 280 285Ala Met Gly Ala Ser Met Val Gly Ile Ala Leu Pro Val Leu Arg Pro290 295 300Ala Ala Lys Gly Asp Val Glu Gly Val Ile Arg Ile Ile Lys Gly Tyr305 310 315 320Ala Glu Glu Ile Arg Asn Val Met Phe Leu Val Gly Ala Arg Asn Ile325 330 335Lys Glu Leu Arg Lys Val Pro Leu Val Ile Thr Gly Phe Val Arg Glu340 345 350Trp Leu Leu Gln Arg Ile Asp Leu Asn Ser Tyr Leu Arg Ala Arg Phe355 360 365Lys Met370<210>69<211>371<212>PRT<213>Pyrococcus horikoshii<400>69Met Lys Glu Glu Leu Thr Ile Leu Arg Lys Phe Glu His Ile Glu His1 5 10 15Cys Leu Lys Arg Asn Val Glu Ala His Val Ser Asn Gly Phe Glu Asp20 25 30Val Tyr Phe Val His Lys Ser Leu Pro Glu Ile Asp Lys Asp Glu Ile35 40 45Asp Leu Thr Val Glu Phe Leu Gly Arg Lys Phe Asp Tyr Pro Ile Met50 55 60Ile Thr Gly Met Thr Gly Gly Thr Arg Arg Glu Glu Ile Ala Gly Lys
65 70 75 80Ile Asn Arg Thr Leu Ala Met Ala Ala Glu Glu Leu Asn Ile Pro Phe85 90 95Gly Val Gly Ser Gln Arg Ala Met Ile Glu Lys Pro Glu Thr Trp Glu100 105 110Ser Tyr Tyr Val Arg Asp Val Ala Pro Asp Ile Phe Leu Ile Gly Asn115 120 125Leu Gly Ala Pro Gln Phe Gly Lys Asn Ala Lys Lys Arg Tyr Ser Val130 135 140Lys Glu Val Leu Tyr Ala Ile Glu Lys Ile Glu Ala Asp Ala Ile Ala145 150 155 160Ile His Met Asn Pro Leu Gln Glu Ser Val Gln Pro Glu Gly Asp Thr165 170 175Thr Tyr Ala Gly Val Leu Glu Ala Leu Ala Glu Ile Lys Ser Ser Ile180 185 190Asn Tyr Pro Val Ile Ala Lys Glu Thr Gly Ala Gly Val Ser Lys Glu195 200 205Val Ala Ile Glu Leu Glu Ser Val Gly Ile Asp Ala Ile Asp Ile Ser210 215 220Gly Leu Gly Gly Thr Ser Trp Ser Ala Val Glu Tyr Tyr Arg Ala Lys225 230 235 240Asp Ser Glu Lys Arg Lys Ile Ala Leu Lys Phe Trp Asp Trp Gly Ile245 250 255Lys Thr Ala Ile Ser Leu Ala Glu Val Arg Trp Ala Thr Asn Leu Pro260 265 270Ile Ile Ala Ser Gly Gly Met Arg Asp Gly Val Met Met Ala Lys Ala275 280 285Leu Ala Met Gly Ala Ser Leu Val Gly Ile Ala Leu Pro Val Leu Arg290 295 300Pro Ala Ala Arg Gly Asp Val Glu Gly Val Val Arg Ile Ile Arg Gly305 310 315 320Tyr Ala Glu Glu Ile Lys Asn Val Met Phe Leu Val Gly Ala Arg Asn325 330 335Ile Arg Glu Leu Arg Arg Val Pro Leu Val Ile Thr Gly Phe Val Arg340 345 350Glu Trp Leu Leu Gln Arg Ile Asp Leu Asn Ser Tyr Leu Arg Ser Arg355 360 365Phe Lys His370
<211>349<212>PRT<213>嗜热碱甲烷杆菌(Methanobacterium thermoautotrophicum)<400>70Met Ile Ser Asp Arg Lys Leu Glu His Leu Ile Leu Cys Ala Ser Cys1 5 10 15Asp Val Glu Tyr Arg Lys Lys Thr Gly Phe Glu Asp Ile Glu Ile Val20 25 30His Arg Ala Ile Pro Glu Ile Asn Lys Glu Lys Ile Asp Ile Ser Leu35 40 45Asp Phe Leu Gly Arg Glu Leu Ser Ser Pro Val Met Ile Ser Ala Ile50 55 60Thr Gly Gly His Pro Ala Ser Met Lys Ile Asn Arg Glu Leu Ala Arg65 70 75 80Ala Ala Glu Lys Leu Gly Ile Ala Leu Gly Leu Gly Ser Gln Arg Ala85 90 95Gly Val Glu His Pro Glu Leu Glu Gly Thr Tyr Thr Ile Ala Arg Glu100 105 110Glu Ala Pro Ser Ala Met Leu Ile Gly Asn Ile Gly Ser Ser His Ile115 120 125Glu Tyr Ala Glu Arg Ala Val Glu Met Ile Asp Ala Asp Ala Leu Ala130 135 140Val His Leu Asn Pro Leu Gln Glu Ser Ile Gln Pro Gly Gly Asp Val145 150 155 160Asp Ser Ser Gly Ala Leu Glu Ser Ile Ser Ala Ile Val Glu Ser Val165 170 175Asp Val Pro Val Met Val Lys Glu Thr Gly Ala Gly Ile Cys Ser Glu180 185 190Asp Ala Ile Glu Leu Glu Ser Cys Gly Val Ser Ala Ile Asp Val Ala195 200 205Gly Ala Gly Gly Thr Ser Trp Ala Ala Val Glu Thr Tyr Arg Ala Asp210 215 220Asp Arg Tyr Leu Gly Glu Leu Phe Trp Asp Trp Gly Ile Pro Thr Ala225 230 235 240Ala Ser Thr Val Glu Val Val Glu Ser Val Ser Ile Pro Val Ile Ala245 250 255Ser Gly Gly Ile Arg Ser Gly Ile Asp Ala Ala Lys Ala Ile Ser Leu260 265 270Gly Ala Glu Met Val Gly Ile Ala Leu Pro Val Leu Glu Ala Ala Gly275 280 285His Gly Tyr Arg Glu Val Ile Lys Val Ile Glu Gly Phe Asn Glu Ala290 295 300
Leu Arg Thr Ala Met Tyr Leu Ala Gly Ala Glu Thr Leu Asp Asp Leu305 310 315 320Lys Lys Ser Pro Val Ile Ile Thr Gly His Thr Gly Glu Trp Leu Asn325 330 335Gln Arg Gly Phe Glu Thr Lys Lys Tyr Ala Arg Arg Ser340 345<210>71<211>359<212>PRT<213>詹氏甲烷球菌(Methanococcus jannaschii)<400>71Met Val Asn Asn Arg Asn Glu Ile Glu Val Arg Lys Leu Glu His Ile1 5 10 15Phe Leu Cys Ser Tyr Cys Asn Val Glu Tyr Glu Lys Thr Thr Leu Leu20 25 30Glu Asp Ile Glu Leu Ile His Lys Gly Thr Cys Gly Ile Asn Phe Asn35 40 45Asp Ile Glu Thr Glu Ile Glu Leu Phe Gly Lys Lys Leu Ser Ala Pro50 55 60Ile Ile Val Ser Gly Met Thr Gly Gly His Ser Lys Ala Lys Glu Ile65 70 75 80Asn Lys Asn Ile Ala Lys Ala Val Glu Glu Leu Gly Leu Gly Met Gly85 90 95Val Gly Ser Gln Arg Ala Ala Ile Val Asn Asp Glu Leu Ile Asp Thr100 105 110Tyr Ser Ile Val Arg Asp Tyr Thr Asn Asn Leu Val Ile Gly Asn Leu115 120 125Gly Ala Val Asn Phe Ile Val Asp Asp Trp Asp Glu Glu Ile Ile Asp130 135 140Lys Ala Ile Glu Met Ile Asp Ala Asp Ala Ile Ala Ile His Phe Asn145 150 155 160Pro Leu Gln Glu Ile Ile Gln Pro Glu Gly Asp Leu Asn Phe Lys Asn165 170 175Leu Tyr Lys Leu Lys Glu Ile Ile Ser Asn Tyr Lys Lys Ser Tyr Lys180 185 190Asn Ile Pro Phe Ile Ala Lys Gln Val Gly Glu Gly Phe Ser Lys Glu195 200 205Asp Ala Leu Ile Leu Lys Asp Ile Gly Phe Asp Ala Ile Asp Val Gln210 215 220Gly Ser Gly Gly Thr Ser Trp Ala Lys Val Glu Ile Tyr Arg Val Lys225 230 235 240
Glu Glu Glu Ile Lys Arg Leu Ala Glu Lys Phe Ala Asn Trp Gly Ile245 250 255Pro Thr Ala Ala Ser Ile Phe Glu Val Lys Ser Val Tyr Asp Gly Ile260 265 270Val Ile Gly Ser Gly Gly Ile Arg Gly Gly Leu Asp Ile Ala Lys Cys275 280 285Ile Ala Ile Gly Cys Asp Cys Cys Ser Val Ala Leu Pro Ile Leu Lys290 295 300Ala Ser Leu Lys Gly Trp Glu Glu Val Val Lys Val Leu Glu Ser Tyr305 310 315 320Ile Lys Glu Leu Lys Ile Ala Met Phe Leu Val Gly Ala Glu Asn Ile325 330 335Glu Glu Leu Lys Lys Thr Ser Tyr Ile Val Lys Gly Thr Leu Lys Glu340 345 350Trp Ile Ser Gln Arg Leu Lys355<210>72<211>348<212>PRT<213>嗜酸热原体(Thermoplasma acidophilum)<400>72Met Ile Gly Lys Arg Lys Glu Glu His Ile Arg Ile Ala Glu Asn Glu1 5 10 15Asp Val Ser Ser Phe His Asn Phe Trp Asp Asp Ile Ser Leu Met His20 25 30Glu Ala Asp Pro Glu Val Asn Tyr Asp Glu Ile Asp Thr Ser Val Asp35 40 45Phe Leu Gly Lys Lys Leu Lys Phe Pro Met Ile Ile Ser Ser Met Thr50 55 60Gly Gly Ala Glu Ile Ala Lys Asn Ile Asn Arg Asn Leu Ala Val Ala65 70 75 80Ala Glu Arg Phe Gly Ile Gly Met Gly Val Gly Ser Met Arg Ala Ala85 90 95Ile Val Asp Arg Ser Ile Glu Asp Thr Tyr Ser Val Ile Asn Glu Ser100 105 110His Val Pro Leu Lys Ile Ala Asn Ile Gly Ala Pro Gln Leu Val Arg115 120 125Gln Asp Lys Asp Ala Val Ser Asn Arg Asp Ile Ala Tyr Ile Tyr Asp130 135 140Leu Ile Lys Ala Asp Phe Leu Ala Val His Phe Asn Phe Leu Gln Glu145 150 155 160Met Val Gln Pro Glu Gly Asp Arg Asn Ser Lys Gly Val Ile Asp Arg165 170 175
Ile Lys Asp Leu Ser Gly Ser Phe Asn Ile Ile Ala Lys Glu Thr Gly180 185 190Ser Gly Phe Ser Arg Arg Thr Ala Glu Arg Leu Ile Asp Ala Gly Val195 200 205Lys Ala Ile Glu Val Ser Gly Val Ser Gly Thr Thr Phe Ala Ala Val210 215 220Glu Tyr Tyr Arg Ala Arg Lys Glu Asn Asn Leu Glu Lys Met Arg Ile225 230 235 240Gly Glu Thr Phe Trp Asn Trp Gly Ile Pro Ser Pro Ala Ser Val Tyr245 250 255Tyr Cys Ser Asp Leu Ala Pro Val Ile Gly Ser Gly Gly Leu Arg Asn260 265 270Gly Leu Asp Leu Ala Lys Ala Ile Ala Met Gly Ala Thr Ala Gly Gly275 280 285Phe Ala Arg Ser Leu Leu Lys Asp Ala Asp Thr Asp Pro Glu Met Leu290 295 300Met Lys Asn Ile Glu Leu Ile Gln Arg Glu Phe Arg Val Ala Leu Phe305 310 315 320Leu Thr Gly Asn Lys Asn Val Tyr Glu Leu Lys Phe Thr Lys Lys Val325 330 335Ile Val Asp Pro Leu Arg Ser Trp Leu Glu Ala Lys340 345<210>73<211>357<212>PRT<213>Leishmania major<400>73Met Ser Ser Arg Asp Cys Thr Val Asp Arg Glu Ala Ala Val Gln Lys1 5 10 15Arg Lys Lys Asp His Ile Asp Ile Cys Leu His Gln Asp Val Glu Pro20 25 30His Lys Arg Arg Thr Ser Ile Trp Asn Lys Tyr Thr Leu Pro Tyr Lys35 40 45Ala Leu Pro Glu Val Asp Leu Gln Lys Ile Asp Thr Ser Cys Glu Phe50 55 60Met Gly Lys Arg Ile Ser Phe Pro Phe Phe Ile Ser Ser Met Thr Gly65 70 75 80Gly Glu Ala His Gly Arg Val Ile Asn Glu Asn Leu Ala Lys Ala Cys85 90 95Glu Ala Glu Lys Ile Pro Phe Gly Leu Gly Ser Met Arg Ile Ile Asn100 105 110
Arg Tyr Ala Ser Ala Val His Thr Phe Asn Val Lys Glu Phe Cys Pro115 120 125Ser Val Pro Met Leu Ala Asn Ile Gly Leu Val Gln Leu Asn Tyr Gly130 135 140Phe Gly Pro Lys Glu Val Asn Asn Leu Val Asn Ser Val Arg Ala Asp145 150 155 160Gly Leu Cys Ile His Leu Asn His Thr Gln Glu Val Cys Gln Pro Glu165 170 175Gly Asp Thr Asn Phe Glu Gly Leu Ile Glu Lys Leu Arg Gln Leu Leu180 185 190Pro His Ile Lys Val Pro Val Leu Val Lys Gly Val Gly His Gly Ile195 200 205Asp Tyr Glu Ser Met Val Ala Ile Lys Ala Ser Gly Val Lys Tyr Val210 215 220Asp Val Ser Gly Cys Gly Gly Thr Ser Trp Ala Trp Ile Glu Gly Arg225 230 235 240Arg Gln Pro Tyr Lys Ala Glu Glu Glu Asn Ile Gly Tyr Leu Leu Arg245 250 255Asp Ile Gly Val Pro Thr Asp Val Cys Leu Arg Glu Ser Ala Pro Leu260 265 270Thr Val Asn Gly Asp Leu His Leu Ile Ala Gly Gly Gly Ile Arg Asn275 280 285Gly Met Asp Val Ala Lys Ala Leu Met Met Gly Ala Glu Tyr Ala Thr290 295 300Ala Ala Met Pro Phe Leu Ala Ala Ala Leu Glu Ser Ser Glu Ala Val305 310 315 320Arg Ala Val Ile Gln Arg Met Arg Gln Glu Leu Arg Val Ser Met Phe325 330 335Thr Cys Gly Ala Arg Asn Ile Glu Glu Leu Arg Arg Met Lys Val Ile340 345 350Glu Leu Gly His Leu355<210>74<211>398<212>PRT<213>肺炎链球菌(Streptococcus pneumoniae)<400>74Met Asn Asp Lys Thr Glu Val Asn Met Thr Ile Gly Ile Asp Lys Ile1 5 10 15Gly Phe Ala Thr Ser Gln Tyr Val Leu Lys Leu Gln Asp Leu Ala Glu20 25 30
Ala Arg Gly Ile Asp Pro Glu Lys Leu Ser Lys Gly Leu Leu Leu Lys35 40 45Glu Leu Ser Ile Ala Pro Leu Thr Glu Asp Ile Val Thr Leu Ala Ala50 55 60Ser Ala Ser Asp Ser Ile Leu Thr Glu Gln Glu Arg Gln Glu Val Asp65 70 75 80Met Val Ile Val Ala Thr Glu Ser Gly Ile Asp Gln Ser Lys Ala Ala85 90 95Ala Val Phe Val His Gly Leu Leu Gly Ile Gln Pro Phe Ala Arg Ser100 105 110Phe Glu Ile Lys Glu Ala Cys Tyr Gly Ala Thr Ala Ala Leu His Tyr115 120 125Ala Lys Leu His Val Glu Asn Ser Pro Glu Ser Lys Val Leu Val Ile130 135 140Ala Ser Asp Ile Ala Lys Tyr Gly Ile Glu Thr Pro Gly Glu Pro Thr145 150 155 160Gln Gly Ala Gly Ser Val Ala Met Leu Ile Thr Gln Asn Pro Arg Met165 170 175Met Ala Phe Asn Asn Asp Asn Val Ala Gln Thr Arg Asp Ile Met Asp180 185 190Phe Trp Arg Pro Asn Tyr Ser Thr Thr Pro Tyr Val Asn Gly Val Tyr195 200 205Ser Thr Gln Gln Tyr Leu Asp Ser Leu Lys Thr Thr Trp Leu Glu Tyr210 215 220Gln Lys Arg Tyr Gln Leu Thr Leu Asp Asp Phe Ala Ala Val Cys Phe225 230 235 240His Leu Pro Tyr Pro Lys Leu Ala Leu Lys Gly Leu Lys Lys Ile Met245 250 255Asp Lys Asn Leu Pro Gln Glu Lys Lys Asp Leu Leu Gln Lys His Phe260 265 270Asp Gln Ser Ile Leu Tyr Ser Gln Lys Val Gly Asn Ile Tyr Thr Gly275 280 285Ser Leu Phe Leu Gly Leu Leu Ser Leu Leu Glu Asn Thr Asp Ser Leu290 295 300Lys Ala Gly Asp Lys Ile Ala Leu Tyr Ser Tyr Gly Ser Gly Ala Val305 310 315 320Ala Glu Phe Phe Ser Gly Glu Leu Val Glu Gly Tyr Glu Ala Tyr Leu325 330 335Asp Lys Asp Arg Leu Asn Lys Leu Asn Gln Arg Thr Ala Leu Ser Val340 345 350Ala Asp Tyr Glu Lys Val Phe Phe Glu Glu Val Asn Leu Asp Glu Thr
355 360 365Asn Ser Ala Gln Phe Ala Gly Tyr Glu Asn Gln Asp Phe Ala Leu Val370 375 380Glu Ile Leu Asp His Gln Arg Arg Tyr Ser Lys Val Glu Lys385 390 395<210>75<211>391<212>PRT<213>酿脓链球菌(streptococcus pyogenes)<400>75Met Thr Ile Gly Ile Asp Lys Ile Gly Phe Ala Thr Ser Gln Tyr Val1 5 10 15Leu Lys Leu Glu Asp Leu Ala Leu Ala Arg Gln Val Asp Pro Ala Lys20 25 30Phe Ser Gln Gly Leu Leu Ile Glu Ser Phe Ser Val Ala Pro Ile Thr35 40 45Glu Asp Ile Ile Thr Leu Ala Ala Ser Ala Ala Asp Gln Ile Leu Thr50 55 60Asp Glu Asp Arg Ala Lys Ile Asp Met Val Ile Leu Ala Thr Glu Ser65 70 75 80Ser Thr Asp Gln Ser Lys Ala Ser Ala Ile Tyr Val His His Leu Val85 90 95Gly Ile Gln Pro Phe Ala Arg Ser Phe Glu Val Lys Gln Ala Cys Tyr100 105 110Ser Ala Thr Ala Ala Leu Asp Tyr Ala Lys Leu His Val Ala Ser Lys115 120 125Pro Asp Ser Arg Val Leu Val Ile Ala Ser Asp Ile Ala Arg Tyr Gly130 135 140Val Gly Ser Pro Gly Glu Ser Thr Gln Gly Ser Gly Ser Ile Ala Leu145 150 155 160Leu Val Thr Ala Asp Pro Arg Ile Leu Ala Leu Asn Glu Asp Asn Val165 170 175Ala Gln Thr Arg Asp Ile Met Asp Phe Trp Arg Pro Asn Tyr Ser Phe180 185 190Thr Pro Tyr Val Asp Gly Ile Tyr Ser Thr Lys Gln Tyr Leu Asn Cys195 200 205Leu Glu Thr Thr Trp Gln Ala Tyr Gln Lys Arg Glu Asn Leu Gln Leu210 215 220Ser Asp Phe Ala Ala Val Cys Phe His Ile Pro Phe Pro Lys Leu Ala225 230235 240Leu Lys Gly Leu Asn Asn Ile Met Asp Asn Thr Val Pro Pro Glu His245 250 255
Arg Glu Lys Leu Ile Glu Ala Phe Gln Ala Ser Ile Thr Tyr Ser Lys260 265 270Gln Ile Gly Asn Ile Tyr Thr Gly Ser Leu Tyr Leu Gly Leu Leu Ser275 280 285Leu Leu Glu Asn Ser Lys Val Leu Gln Ser Gly Asp Lys Ile Gly Phe290 295 300Phe Ser Tyr Gly Ser Gly Ala Val Ser Glu Phe Tyr Ser Gly Gln Leu305 310 315 320Val Ala Gly Tyr Asp Lys Met Leu Met Thr Asn Arg Gln Ala Leu Leu325 330 335Asp Gln Arg Thr Arg Leu Ser Val Ser Lys Tyr Glu Asp Leu Phe Tyr340 345 350Glu Gln Val Gln Leu Asp Asp Asn Gly Asn Ala Asn Phe Asp Ile Tyr355 360 365Leu Thr Gly Lys Phe Ala Leu Thr Ala Ile Lys Glu His Gln Arg Ile370 375 380Tyr His Thr Asn Asp Lys Asn385 390<210>76<211>384<212>PRT<213>粪肠球菌(Enterococcus faecalis)<400>76Glu Met Thr Ile Gly Ile Asp Lys Ile Ser Phe Phe Val Pro Pro Tyr1 5 10 15Tyr Ile Asp Met Thr Ala Leu Ala Glu Ala Arg Asn Val Asp Pro Gly20 25 30Lys Phe His Ile Gly Ile Gly Gln Asp Gln Met Ala Val Asn Pro Ile35 40 45Ser Gln Asp Ile Val Thr Phe Ala Ala Asn Ala Ala Glu Ala Ile Leu50 55 60Thr Lys Glu Asp Lys Glu Ala Ile Asp Met Val Ile Val Gly Thr Glu65 70 75 80Ser Ser Ile Asp Glu Ser Lys Ala Ala Ala Val Val Leu His Arg Leu85 90 95Met Gly Ile Gln Pro Phe Ala Arg Ser Phe Glu Ile Lys Glu Ala Cys100 105 110Tyr Gly Ala Thr Ala Gly Leu Gln Leu Ala Lys Asn His Val Ala Leu115 120 125His Pro Asp Lys Lys Val Leu Val Val Ala Ala Asp Ile Ala Lys Tyr130 135 140Gly Leu Asn Ser Gly Gly Glu Pro Thr Gln Gly Ala Gly Ala Val Ala145 150 155 160
Met Leu Val Ala Ser Glu Pro Arg Ile Leu Ala Leu Lys Glu Asp Asn165 170 175Val Met Leu Thr Gln Asp Ile Tyr Asp Phe Trp Arg Pro Thr Gly His180 185 190Pro Tyr Pro Met Val Asp Gly Pro Leu Ser Asn Glu Thr Tyr Ile Gln195 200 205Ser Phe Ala Gln Val Trp Asp Glu His Lys Lys Arg Thr Gly Leu Asp210 215 220Phe Ala Asp Tyr Asp Ala Leu Ala Phe His Ile Pro Tyr Thr Lys Met225 230 235 240Gly Lys Lys Ala Leu Leu Ala Lys Ile Ser Asp Gln Thr Glu Ala Glu245 250 255Gln Glu Arg Ile Leu Ala Arg Tyr Glu Glu Ser Ile Ile Tyr Ser Arg260 265 270Arg Val Gly Asn Leu Tyr Thr Gly Ser Leu Tyr Leu Gly Leu Ile Ser275 280 285Leu Leu Glu Asn Ala Thr Thr Leu Thr Ala Gly Asn Gln Ile Gly Leu290 295 300Phe Ser Tyr Gly Ser Gly Ala Val Ala Glu Phe Phe Thr Gly Glu Leu305 310 315 320Val Ala Gly Tyr Gln Asn His Leu Gln Lys Glu Thr His Leu Ala Leu325 330 335Leu Asp Asn Arg Thr Glu Leu Ser Ile Ala Glu Tyr Glu Ala Met Phe340 345 350Ala Glu Thr Leu Asp Thr Asp Ile Asp Gln Thr Leu Glu Asp Glu Leu355 360 365Lys Tyr Ser Ile Ser Ala Ile Asn Asn Thr Val Arg Ser Tyr Arg Asn370 375 380<210>77<211>384<212>PRT<213>屎肠球菌(Enterococcus faecium)<400>77Met Lys Ile Gly Ile Asp Arg Leu Ser Phe Phe Ile Pro Asn Leu Tyr1 5 10 15Leu Asp Met Thr Glu Leu Ala Glu Ser Arg Gly Asp Asp Pro Ala Lys20 25 30Tyr His Ile Gly Ile Gly Gln Asp Gln Met Ala Val Asn Arg Ala Asn35 40 45Glu Asp Ile Ile Thr Leu Gly Ala Asn Ala Ala Ser Lys Ile Val Thr50 55 60
Glu Lys Asp Arg Glu Leu Ile Asp Met Val Ile Val Gly Thr Glu Ser65 70 75 80Gly Ile Asp His Ser Lys Ala Ser Ala Val Ile Ile His His Leu Leu85 90 95Lys Ile Gln Ser Phe Ala Arg Ser Phe Glu Val Lys Glu Ala Cys Tyr100 105 110Gly Gly Thr Ala Ala Leu His Met Ala Lys Glu Tyr Val Lys Asn His115 120 125Pro Glu Arg Lys Val Leu Val Ile Ala Ser Asp Ile Ala Arg Tyr Gly130 135 140Leu Ala Ser Gly Gly Glu Val Thr Gln Gly Val Gly Ala Val Ala Met145 150 155 160Met Ile Thr Gln Asn Pro Arg Ile Leu Ser Ile Glu Asp Asp Ser Val165 170 175Phe Leu Thr Glu Asp Ile Tyr Asp Phe Trp Arg Pro Asp Tyr Ser Glu180 185 190Phe Pro Val Val Asp Gly Pro Leu Ser Asn Ser Thr Tyr Ile Glu Ser195 200 205Phe Gln Lys Val Trp Asn Arg His Lys Glu Leu Ser Gly Arg Gly Leu210 215 220Glu Asp Tyr Gln Ala Ile Ala Phe His Ile Pro Tyr Thr Lys Met Gly225 230 235 240Lys Lys Ala Leu Gln Ser Val Leu Asp Gln Thr Asp Glu Asp Asn Gln245 250 255Glu Arg Leu Met Ala Arg Tyr Glu Glu Ser Ile Arg Tyr Ser Arg Arg260 265 270Ile Gly Asn Leu Tyr Thr Gly Ser Leu Tyr Leu Gly Leu Thr Ser Leu275 280 285Leu Glu Asn Ser Lys Ser Leu Gln Pro Gly Asp Arg Ile Gly Leu Phe290 295 300Ser Tyr Gly Ser Gly Ala Val Ser Glu Phe Phe Thr Gly Tyr Leu Glu305 310 315 320Glu Asn Tyr Gln Glu Tyr Leu Phe Ala Gln Ser His Gln Glu Met Leu325 330 335Asp Ser Arg Thr Arg Ile Thr Val Asp Glu Tyr Glu Thr Ile Phe Ser340 345 350Glu Thr Leu Pro Glu His Gly Glu Cys Ala Glu Tyr Thr Ser Asp Val355 360 365Pro Phe Ser Ile Thr Lys Ile Glu Asn Asp Ile Arg Tyr Tyr Lys Ile370 375 380
<210>78<211>388<212>PRT<213>溶血葡萄球菌(Staphylococcus haemolyticus)<400>78Met Ser Ile Gly Ile Asp Lys Ile Asn Phe Tyr Val Pro Lys Tyr Tyr1 5 10 15Val Asp Met Ala Lys Leu Ala Glu Ala Arg Gln Val Asp Pro Asn Lys20 25 30Phe Leu Ile Gly Ile Gly Gln Thr Gln Met Ala Val Ser Pro Val Ser35 40 45Gln Asp Ile Val Ser Met Gly Ala Asn Ala Ala Lys Asp Ile Ile Thr50 55 60Asp Asp Asp Lys Lys His Ile Gly Met Val Ile Val Ala Thr Glu Ser65 70 75 80Ala Ile Asp Asn Ala Lys Ala Ala Ala Val Gln Ile His Asn Leu Leu85 90 95Gly Val Gln Pro Phe Ala Arg Cys Phe Glu Met Lys Glu Ala Cys Tyr100 105 110Ala Ala Thr Pro Ala Ile Gln Leu Ala Lys Asp Tyr Ile Glu Lys Arg115 120 125Pro Asn Glu Lys Val Leu Val Ile Ala Ser Asp Thr Ala Arg Tyr Gly130 135 140Ile Gln Ser Gly Gly Glu Pro Thr Gln Gly Ala Gly Ala Val Ala Met145 150 155 160Leu Ile Ser Asn Asn Pro Ser Ile Leu Glu Leu Asn Asp Asp Ala Val165 170 175Ala Tyr Thr Glu Asp Val Tyr Asp Phe Trp Arg Pro Thr Gly His Lys180 185 190Tyr Pro Leu Val Ala Gly Ala Leu Ser Lys Asp Ala Tyr Ile Lys Ser195 200 205Phe Gln Glu Ser Trp Asn Glu Tyr Ala Arg Arg Glu Asp Lys Thr Leu210 215 220Ser Asp Phe Glu Ser Leu Cys Phe His Val Pro Phe Thr Lys Met Gly225 230 235 240Lys Lys Ala Leu Asp Ser Ile Ile Asn Asp Ala Asp Glu Thr Thr Gln245 250 255Glu Arg Leu Thr Ser Gly Tyr Glu Asp Ala Val Tyr Tyr Asn Arg Tyr260 265 270Val Gly Asn Ile Tyr Thr Gly Ser Leu Tyr Leu Ser Leu Ile Ser Leu275 280 285Leu Glu Asn Arg Ser Leu Lys Gly Gly Gln Thr Ile Gly Leu Phe Ser
290 295 300Tyr Gly Ser Gly Ser Val Gly Glu Phe Phe Ser Ala Thr Leu Val Glu305 310 315 320Gly Tyr Glu Lys Gln Leu Asp Ile Glu Gly His Lys Ala Leu Leu Asn325 330 335Glu Arg Gln Glu Val Ser Val Glu Asp Tyr Glu Ser Phe Phe Lys Arg340 345 350Phe Asp Asp Leu Glu Phe Asp His Ala Thr Glu Gln Thr Asp Asp Asp355 360 365Lys Ser Ile Tyr Tyr Leu Glu Asn Ile Gln Asp Asp Ile Arg Gln Tyr370 375 380His Ile Pro Lys385<210>79<211>388<212>PRT<213>表皮葡萄球菌(Staphylococcus epidermidis)<400>79Met Asn Ile Gly Ile Asp Lys Ile Ser Phe Tyr Val Pro Lys Tyr Tyr1 5 10 15Val Asp Met Ala Lys Leu Ala Glu Ala Arg Gln Val Asp Pro Asn Lys20 25 30Phe Leu Ile Gly Ile Gly Gln Thr Glu Met Thr Val Ser Pro Val Asn35 40 45Gln Asp Ile Val Ser Met Gly Ala Asn Ala Ala Lys Asp Ile Ile Thr50 55 60Glu Glu Asp Lys Lys Asn Ile Gly Met Val Ile Val Ala Thr Glu Ser65 70 75 80Ala Ile Asp Asn Ala Lys Ala Ala Ala Val Gln Ile His His Leu Leu85 90 95Gly Ile Gln Pro Phe Ala Arg Cys Phe Glu Met Lys Glu Ala Cys Tyr100 105 110Ala Ala Thr Pro Ala Ile Gln Leu Ala Lys Asp Tyr Leu Ala Gln Arg115 120 125Pro Asn Glu Lys Val Leu Val Ile Ala Ser Asp Thr Ala Arg Tyr Gly130 135 140Ile His Ser Gly Gly Glu Pro Thr Gln Gly Ala Gly Ala Val Ala Met145 150 155 160Met Ile Ser His Asp Pro Ser Ile Leu Lys Leu Asn Asp Asp Ala Val165 170 175Ala Tyr Thr Glu Asp Val Tyr Asp Phe Trp Arg Pro Thr Gly His Gln180 185 190
Tyr Pro Leu Val Ala Gly Ala Leu Ser Lys Asp Ala Tyr Ile Lys Ser195 200 205Phe Gln Glu Ser Trp Asn Glu Tyr Ala Arg Arg His Asn Lys Thr Leu210 215 220Ala Asp Phe Ala Ser Leu Cys Phe His Val Pro Phe Thr Lys Met Gly225 230 235 240Gln Lys Ala Leu Asp Ser Ile Ile Asn His Ala Asp Glu Thr Thr Gln245 250 255Asp Arg Leu Asn Ser Ser Tyr Gln Asp Ala Val Asp Tyr Asn Arg Tyr260 265 270Val Gly Asn Ile Tyr Thr Gly Ser Leu Tyr Leu Ser Leu Ile Ser Leu275 280 285Leu Glu Thr Arg Asp Leu Lys Gly Gly Gln Thr Ile Gly Leu Phe Ser290 295 300Tyr Gly Ser Gly Ser Val Gly Glu Phe Phe Ser Gly Thr Leu Val Asp305 310 315 320Gly Phe Lys Glu Gln Leu Asp Val Glu Arg His Lys Ser Leu Leu Asn325 330 335Asn Arg Ile Glu Val Ser Val Asp Glu Tyr Glu His Phe Phe Lys Arg340 345 350Phe Asp Gln Leu Glu Leu Asn His Glu Leu Glu Lys Ser Asn Ala Asp355 360 365Arg Asp Ile Phe Tyr Leu Lys Ser Ile Asp Asn Asn Ile Arg Glu Tyr370 375 380His Ile Ala Glu385<210>80<211>388<212>PRT<213>金黄色葡萄球菌(Staphylococcus aureus)<400>80Met Thr Ile Gly Ile Asp Lys Ile Asn Phe Tyr Val Pro Lys Tyr Tyr1 5 10 15Val Asp Met Ala Lys Leu Ala Glu Ala Arg Gln Val Asp Pro Asn Lys20 25 30Phe Leu Ile Gly Ile Gly Gln Thr Glu Met Ala Val Ser Pro Val Asn35 40 45Gln Asp Ile Val Ser Met Gly Ala Asn Ala Ala Lys Asp Ile Ile Thr50 55 60Asp Glu Asp Lys Lys Lys Ile Gly Met Val Ile Val Ala Thr Glu Ser65 70 75 80
Ala Val Asp Ala Ala Lys Ala Ala Ala Val Gln Ile His Asn Leu Leu85 90 95Gly Ile Gln Pro Phe Ala Arg Cys Phe Glu Met Lys Glu Ala Cys Tyr100 105 110Ala Ala Thr Pro Ala Ile Gln Leu Ala Lys Asp Tyr Leu Ala Thr Arg115 120 125Pro Asn Glu Lys Val Leu Val Ile Ala Thr Asp Thr Ala Arg Tyr Gly130 135 140Leu Asn Ser Gly Gly Glu Pro Thr Gln Gly Ala Gly Ala Val Ala Met145 150 155 160Val Ile Ala His Asn Pro Ser Ile Leu Ala Leu Asn Glu Asp Ala Val165 170 175Ala Tyr Thr Glu Asp Val Tyr Asp Phe Trp Arg Pro Thr Gly His Lys180 185 190Tyr Pro Leu Val Asp Gly Ala Leu Ser Lys Asp Ala Tyr Ile Arg Ser195 200 205Phe Gln Gln Ser Trp Asn Glu Tyr Ala Lys Arg Gln Gly Lys Ser Leu210 215 220Ala Asp Phe Ala Ser Leu Cys Phe His Val Pro Phe Thr Lys Met Gly225 230 235 240Lys Lys Ala Leu Glu Ser Ile Ile Asp Asn Ala Asp Glu Thr Thr Gln245 250 255Glu Arg Leu Arg Ser Gly Tyr Glu Asp Ala Val Asp Tyr Asn Arg Tyr260 265 270Val Gly Asn Ile Tyr Thr Gly Ser Leu Tyr Leu Ser Leu Ile Ser Leu275 280 285Leu Glu Asn Arg Asp Leu Gln Ala Gly Glu Thr Ile Gly Leu Phe Ser290 295 300Tyr Gly Ser Gly Ser Val Val Glu Phe Tyr Ser Ala Thr Leu Val Val305 310 315 320Gly Tyr Lys Asp His Leu Asp Gln Ala Ala His Lys Ala Leu Leu Asn325 330 335Asn Arg Thr Glu Val Ser Val Asp Ala Tyr Glu Thr Phe Phe Lys Arg340 345 350Phe Asp Asp Val Glu Phe Asp Glu Glu Gln Asp Ala Val His Glu Asp355 360 365Arg His Ile Phe Tyr Leu Ser Asn Ile Glu Asn Asn Val Arg Glu Tyr370 375 380His Arg Pro Glu385
<210>81<211>389<212>PRT<213>肉葡萄球菌(Staphylococcus carnosus)<400>81Met Thr Ile Gly Ile Asp Gln Leu Asn Phe Tyr Ile Pro Asn Phe Tyr1 5 10 15Val Asp Met Ala Glu Leu Ala Glu Ala Arg Gly Val Asp Pro Asn Lys20 25 30Phe Leu Ile Gly Ile Gly Gln Ser Gln Met Ala Val Ser Pro Val Ser35 40 45Gln Asp Ile Val Ser Met Gly Ala Asn Ala Ala Gln Pro Ile Leu Ser50 55 60Glu Gln Asp Lys Lys Asp Ile Thr Met Val Ile Val Ala Thr Glu Ser65 70 75 80Ala Ile Asp Ser Ala Lys Ala Ser Ala Val Gln Ile His His Leu Leu85 90 95Gly Ile Gln Pro Phe Ala Arg Cys Phe Glu Met Lys Glu Ala Cys Tyr100 105 110Ala Ala Thr Pro Ala Ile Gln Leu Ala Lys Asp Tyr Leu Val Pro Arg115 120 125Pro Lys Glu Lys Val Leu Val Ile Ala Ser Asp Thr Ala Arg Tyr Gly130 135 140Leu Asn Ser Gly Gly Glu Pro Thr Gln Gly Ala Gly Ala Val Ala Met145 150 155 160Val Ile Ser His Asn Pro Ser Ile Leu Glu Leu His Asp Asp Ser Val165 170 175Ala Tyr Thr Glu Asp Val Tyr Asp Phe Trp Arg Pro Ser Gly Glu Ile180 185 190Tyr Pro Leu Val Ala Gly Lys Leu Ser Lys Asp Ala Tyr Ile Lys Ser195 200 205Phe Gln Glu Ser Trp Asn Glu Tyr Ala Lys Arg His His Lys Ser Leu210 215 220Ser Asp Phe Ala Ala Leu Cys Phe His Val Pro Phe Thr Lys Met Gly225 230 235 240Gln Lys Ala Leu Asp Ser Ile Leu Thr Asp Ser Ala Ser Glu Asp Thr245 250 255Gln Ala Arg Leu Asn Glu Gly Tyr Lys Ser Ala Thr Asp Tyr Asn Arg260 265 270Tyr Val Gly Asn Val Tyr Thr Gly Ser Leu Tyr Leu Ser Leu Ile Ser275 280 285Leu Leu Glu Asn His Lys Leu Asn Gly Gly Asp Asn Ile Gly Leu Phe290 295 300
Ser Tyr Gly Ser Gly Ser Val Gly Glu Phe Phe Ser Ala Thr Leu Val305 310 315 320Asp Asn Tyr Gln Asp His Leu Asp Val Lys Ala His Lys Ala Met Leu325 330 335Asp Asn Arg Lys Ala Leu Ser Val Glu Glu Tyr Glu Lys Phe Phe Asn340 345 350Arg Phe Asp Asn Leu Glu Phe Asp Thr Glu Thr Glu Leu Glu Val Glu355 360 365Pro Lys Gly Asn Phe Tyr Leu Lys Glu Ile Ser Asp Asn Ile Arg Tyr370 375 380Tyr Asp Thr Val Lys385<210>82<211>389<212>PRT<213>链霉菌属菌种(Streptomyces sp.)<400>82Met Ser Ile Ser Ile Gly Ile His Asp Leu Ser Phe Ala Thr Thr Glu1 5 10 15Phe Val Leu Pro His Thr Ala Leu Ala Glu Tyr Asn Gly Thr Glu Ile20 25 30Gly Lys Tyr His Val Gly Ile Gly Gln Gln Ser Met Ser Val Pro Ala35 40 45Ala Asp Glu Asp Ile Val Thr Met Ala Ala Thr Ala Ala Arg Pro Ile50 55 60Ile Glu Arg Asn Gly Lys Ser Arg Ile Arg Thr Val Val Phe Ala Thr65 70 75 80Glu Ser Ser Ile Asp Gln Ala Lys Ala Gly Gly Val Tyr Val His Ser85 90 95Leu Leu Gly Leu Glu Ser Ala Cys Arg Val Val Glu Leu Lys Gln Ala100 105 110Cys Tyr Gly Ala Thr Ala Ala Leu Gln Phe Ala Ile Gly Leu Val Arg115 120 125Arg Asp Pro Ala Gln Gln Val Leu Val Ile Ala Ser Asp Val Ser Lys130 135 140Tyr Glu Leu Asp Ser Pro Gly Glu Ala Thr Gln Gly Ala Ala Ala Val145 150 155 160Ala Met Leu Val Gly Ala Asp Pro Ala Leu Leu Arg Ile Glu Glu Pro165 170 175Ser Gly Leu Phe Thr Ala Asp Val Met Asp Phe Trp Arg Pro Asn Tyr180 185 190Leu Thr Thr Ala Leu Val Asp Gly Gln Glu Ser Ile Asn Ala Tyr Leu
195 200 205Gln Ala Val Glu Gly Ala Trp Lys Asp Tyr Ala Glu Gln Asp Gly Arg210 215 220Ser Leu Glu Glu Phe Ala Ala Phe Val Tyr His Gln Pro Phe Thr Lys225 230 235 240Met Ala Tyr Lys Ala His Arg His Leu Leu Asn Phe Asn Gly Tyr Asp245 250 255Thr Asp Lys Asp Ala Ile Glu Gly Ala Leu Gly Gln Thr Thr Ala Tyr260 265 270Asn Asn Val Ile Gly Asn Ser Tyr Thr Ala Ser Val Tyr Leu Gly Leu275 280 285Ala Ala Leu Leu Asp Gln Ala Asp Asp Leu Thr Gly Arg Ser Ile Gly290 295 300Phe Leu Ser Tyr Gly Ser Gly Ser Val Ala Glu Phe Phe Ser Gly Thr305 310 315 320Val Val Ala Gly Tyr Arg Glu Arg Leu Arg Thr Glu Ala Asn Gln Glu325 330 335Ala Ile Ala Arg Arg Lys Ser Val Asp Tyr Ala Thr Tyr Arg Glu Leu340 345 350His Glu Tyr Thr Leu Pro Ser Asp Gly Gly Asp His Ala Thr Pro Val355 360 365Gln Thr Thr Gly Pro Phe Arg Leu Ala Gly Ile Asn Asp His Lys Arg370 375 380Ile Tyr Glu Ala Arg385<210>83<211>389<212>PRT<213>灰孢链霉菌(Streptomyces griseosporeus)<400>83Met Pro Leu Ala Ile Gly Ile His Asp Leu Ser Phe Ala Thr Gly Glu1 5 10 15Phe Val Leu Pro His Thr Ala Leu Ala Ala His Asn Gly Thr Glu Ile20 25 30Gly Lys Tyr His Ala Gly Ile Gly Gln Glu Ser Met Ser Val Pro Ala35 40 45Ala Asp Glu Asp Ile Val Thr Leu Ala Ala Thr Ala Ala Ala Pro Ile50 55 60Val Ala Arg His Gly Ser Asp Arg Ile Arg Thr Val Val Leu Ala Thr65 70 75 80Glu Ser Ser Ile Asp Gln Ala Lys Ser Ala Gly Val Tyr Val His Ser85 90 95
Leu Leu Gly Leu Pro Ser Ala Thr Arg Val Val Glu Leu Lys Gln Ala100 105 110Cys Tyr Gly Ala Thr Ala Gly Leu Gln Phe Ala Ile Gly Leu Val Gln115 120 125Arg Asp Pro Ala Gln Gln Val Leu Val Ile Ala Ser Asp Val Ser Lys130 135 140Tyr Asp Leu Asp Ser Pro Gly Glu Ala Thr Gln Gly Ala Ala Ala Val145 150 155 160Ala Met Leu Val Gly Ala Asp Pro Gly Leu Val Arg Ile Glu Asp Pro165 170 175Ser Gly Leu Phe Thr Val Asp Val Met Asp Phe Trp Arg Pro Asn Tyr180 185 190Arg Thr Thr Ala Leu Val Asp Gly Gln Glu Ser Ile Gly Ala Tyr Leu195 200 205Gln Ala Val Glu Gly Ala Trp Lys Asp Tyr Ser Glu Arg Gly Gly His210 215 220Ser Leu Glu Gln Phe Ala Ala Phe Cys Tyr His Gln Pro Phe Thr Lys225 230 235 240Met Ala His Lys Ala His Arg His Leu Leu Asn Tyr Cys Ser His Asp245 250 255Ile His His Asp Asp Val Thr Arg Ala Val Gly Arg Thr Thr Ala Tyr260 265 270Asn Arg Val Ile Gly Asn Ser Tyr Thr Ala Ser Val Tyr Leu Gly Leu275 280 285Ala Ala Leu Leu Asp Gln Ala Asp Asp Leu Thr Gly Glu Arg Ile Gly290 295 300Phe Leu Ser Tyr Gly Ser Gly Ser Val Ala Glu Phe Phe Gly Gly Ile305 310 315 320Val Val Ala Gly Tyr Arg Asp Arg Leu Arg Thr Ala Ala Asn Ile Glu325 330 335Ala Val Ser Arg Arg Arg Pro Ile Asp Tyr Ala Gly Tyr Arg Glu Leu340 345 350His Glu Trp Ala Phe Pro Ala Arg Arg Gly Ala His Ser Thr Pro Gln355 360 365Gln Thr Thr Gly Pro Phe Arg Leu Ser Gly Ile Ser Gly His Lys Arg370 375 380Leu Tyr Arg Ala Cys385
<210>84<211>407<212>PRT<213>布氏疏螺旋体(Borrelia burgdorferi)<400>84Met Arg Ile Gly Ile Ser Asp Ile Arg Ile Phe Leu Pro Leu Asn Tyr1 5 10 15Leu Asp Phe Ser Val Leu Leu Glu Asn Pro Leu Tyr Phe Ser Asn Glu20 25 30Val Phe Phe Lys Lys Ile Asn Arg Ala Ile Asp Ala Thr Leu Gln Lys35 40 45Gly Phe Arg Phe Thr Ser Pro Asn Glu Asp Ser Val Thr Met Ala Ser50 55 60Ser Ala Val Lys Leu Ile Phe Asp Asn Asn Asn Leu Asp Leu Ser Lys65 70 75 80Ile Arg Ile Leu Leu Gly Gly Thr Glu Thr Gly Val Asp His Ser Lys85 90 95Ala Ile Ser Ser Tyr Val Phe Gly Ala Leu Lys Gln Ser Gly Ile Cys100 105 110Leu Gly Asn Asn Phe Leu Thr Phe Gln Val Gln His Ala Cys Ala Gly115 120 125Ala Ala Met Ser Leu His Thr Val Ala Ser Val Leu Ser His Ser Asn130 135 140Asn Ser Glu Tyr Gly Ile Val Phe Ser Ser Asp Ile Ala His Tyr Ser145 150 155 160Asn Leu Thr Thr Ala Glu Ile Thr Gln Gly Ala Gly Ala Thr Ala Ile165 170 175Leu Ile Glu Lys Asn Pro Lys Ile Leu Ser Ile Asn Leu Ser Glu Phe180 185 190Gly Val Tyr Thr Asp Asp Val Asp Asp Phe Phe Arg Pro Phe Gly Ser195 200 205Val Glu Ala Lys Val Arg Gly Gln Tyr Ser Val Glu Cys Tyr Asn Asn210 215 220Ala Asn Glu Asn Ala Leu Arg Asp Phe Ala Phe Lys Lys Gln Leu Ser225 230 235 240Met Lys Asp Leu Phe Ser Asn Tyr Arg Phe Val Leu His Val Pro Phe245 250 255Ala Lys Met Pro Ile Asp Ser Met His Tyr Ile Leu Lys Lys Tyr Tyr260 265 270Ser Asp Asp Glu Ser Val Arg Asn Ala Tyr Leu Glu Ser Ile Asp Phe275 280 285Tyr Asp Gly Val Glu Ala Ala Met Glu Val Gly Asn Leu Tyr Thr Gly290 295 300
Ser Ile Phe Leu Ser Leu Ala Phe Tyr Leu Lys Arg Val Phe Ser Lys305 310 315 320Lys Asp Ile Thr Gly Glu Lys Ile Leu Phe Cys Ser Tyr Gly Ser Gly325 330 335Asn Ile Met Ile Ile Tyr Glu Leu Thr Ile Glu Lys Ser Ala Phe Asp340 345 350Val Ile Lys Leu Trp Asp Leu Glu Gly Leu Ile Lys Asn Arg Asn Asn355 360 365Ala Asn Phe Glu Glu Tyr Lys Asp Phe Phe Gln Asn Lys Ile Ile Pro370 375 380Gly Glu Ser Arg Gly Phe Tyr Leu Lys Glu Leu Arg Asn Asp Gly Tyr385 390 395 400Arg Val Tyr Gly Tyr Arg Ala405<210>85<211>317<212>PRT<213>肺炎链球菌(Streptococcus pneumoniae)<400>85Met Asp Arg Glu Pro Val Thr Val Arg Ser Tyr Ala Asn Ile Ala Ile1 5 10 15Ile Lys Tyr Trp Gly Lys Lys Lys Glu Lys Glu Met Val Pro Ala Thr20 25 30Ser Ser Ile Ser Leu Thr Leu Glu Asn Met Tyr Thr Glu Thr Thr Leu35 40 45Ser Pro Leu Pro Ala Asn Val Thr Ala Asp Glu Phe Tyr Ile Asn Gly50 55 60Gln Leu Gln Asn Glu Val Glu His Ala Lys Met Ser Lys Ile Ile Asp65 70 75 80Arg Tyr Arg Pro Ala Gly Glu Gly Phe Val Arg Ile Asp Thr Gln Asn85 90 95Asn Met Pro Thr Ala Ala Gly Leu Ser Ser Ser Ser Ser Gly Leu Ser100 105 110Ala Leu Val Lys Ala Cys Asn Ala Tyr Phe Lys Leu Gly Leu Asp Arg115 120 125Ser Gln Leu Ala Gln Glu Ala Lys Phe Ala Ser Gly Ser Ser Ser Arg130 135 140Ser Phe Tyr Gly Pro Leu Gly Ala Trp Asp Lys Asp Ser Gly Glu Ile145 150 155 160Tyr Pro Val Glu Thr Asp Leu Lys Leu Ala Met Ile Met Leu Val Leu165 170 175
Glu Asp Lys Lys Lys Pro Ile Ser Ser Arg Asp Gly Met Lys Leu Cys180 185 190Val Glu Thr Ser Thr Thr Phe Asp Asp Trp Val Arg Gln Ser Glu Lys195 200 205Asp Tyr Gln Asp Met Leu Ile Tyr Leu Lys Glu Asn Asp Phe Ala Lys210 215 220Ile Gly Glu Leu Thr Glu Lys Asn Ala Leu Ala Met His Ala Thr Thr225 230 235 240Lys Thr Ala Ser Pro Ala Phe Ser Tyr Leu Thr Asp Ala Ser Tyr Glu245 250 255Ala Met Ala Phe Val Arg Gln Leu Arg Glu Lys Gly Glu Ala Cys Tyr260 265 270Phe Thr Met Asp Ala Gly Pro Asn Val Lys Val Phe Cys Gln Glu Lys275 280 285Asp Leu Glu His Leu Ser Glu Ile Phe Gly Gln Arg Tyr Arg Leu Ile290 295 300Val Ser Lys Thr Lys Asp Leu Ser Gln Asp Asp Cys Cys305 310 315<210>86<211>314<212>PRT<213>酿脓链球菌(Streptococcus pyogenes)<400>86Met Asp Pro Asn Val Ile Thr Val Thr Ser Tyr Ala Asn Ile Ala Ile1 5 10 15Ile Lys Tyr Trp Gly Lys Glu Asn Gln Ala Lys Met Ile Pro Ser Thr20 25 30Ser Ser Ile Ser Leu Thr Leu Glu Asn Met Phe Thr Thr Thr Ser Val35 40 45Ser Phe Leu Pro Asp Thr Ala Thr Ser Asp Gln Phe Tyr Ile Asn Gly50 55 60Val Leu Gln Asn Asp Glu Glu His Thr Lys Ile Ser Thr Ile Ile Asp65 70 75 80Gln Phe Arg Gln Pro Gly Gln Ala Phe Val Lys Met Glu Thr Gln Asn85 90 95Asn Met Pro Thr Ala Ala Gly Leu Ser Ser Ser Ser Ser Gly Leu Ser100 105 110Ala Leu Val Lys Ala Cys Asp Gln Leu Phe Asp Thr Gln Leu Asp Gln115 120 125Lys Ala Leu Ala Gln Lys Ala Lys Phe Ala Ser Gly Ser Ser Ser Arg130 135 140Ser Phe Phe Gly Pro Val Ala Ala Trp Asp Lys Asp Ser Gly Ala Ile
145 150 155 160Tyr Lys Val Glu Thr Asp Leu Lys Met Ala Met Ile Met Leu Val Leu165 170 175Asn Ala Ala Lys Lys Pro Ile Ser Ser Arg Glu Gly Met Lys Leu Cys180 185 190Arg Asp Thr Ser Thr Thr Phe Asp Glu Trp Val Glu Gln Ser Ala Ile195 200 205Asp Tyr Gln His Met Leu Thr Tyr Leu Lys Thr Asn Asn Phe Glu Lys210 215 220Val Gly Gln Leu Thr Glu Ala Asn Ala Leu Ala Met His Ala Thr Thr225 230 235 240Lys Thr Ala Asn Pro Pro Phe Ser Tyr Leu Thr Lys Glu Ser Tyr Gln245 250 255Ala Met Glu Ala Val Lys Glu Leu Arg Gln Glu Gly Phe Ala Cys Tyr260 265 270Phe Thr Met Asp Ala Gly Pro Asn Val Lys Val Leu Cys Leu Glu Lys275 280 285Asp Leu Ala Gln Leu Ala Glu Arg Leu Gly Lys Asn Tyr Arg Ile Ile290 295 300Val Ser Lys Thr Lys Asp Leu Pro Asp Val305 310<210>87<211>331<212>PRT<213>粪肠球菌(Enterococcus faecalis)<400>87Met Leu Ser Gly Lys Ala Arg Ala His Thr Asn Ile Ala Leu Ile Lys1 5 10 15Tyr Trp Gly Lys Ala Asn Glu Glu Tyr Ile Leu Pro Met Asn Ser Ser20 25 30Leu Ser Leu Thr Leu Asp Ala Phe Tyr Thr Glu Thr Thr Val Thr Phe35 40 45Asp Ala His Tyr Ser Glu Asp Val Phe Ile Leu Asn Gly Ile Leu Gln50 55 60Asn Glu Lys Gln Thr Lys Lys Val Lys Glu Phe Leu Asn Leu Val Arg65 70 75 80Gln Gln Ala Asp Cys Thr Trp Phe Ala Lys Val Glu Ser Gln Asn Phe85 90 95Val Pro Thr Ala Ala Gly Leu Ala Ser Ser Ala Ser Gly Leu Ala Ala100 105 110Leu Ala Gly Ala Cys Asn Val Ala Leu Gly Leu Asn Leu Ser Ala Lys115 120 125Asp Leu Ser Arg Leu Ala Arg Arg Gly Ser Gly Ser Ala Cys Arg Ser
130 135 140Ile Phe Gly Gly Phe Ala Gln Trp Asn Lys Gly His Ser Asp Glu Thr145 150 155 160Ser Phe Ala Glu Asn Ile Pro Ala Asn Asn Trp Glu Asn Glu Leu Ala165 170 175Met Leu Phe Ile Leu Ile Asn Asp Gly Glu Lys Asp Val Ser Ser Arg180 185 190Asp Gly Met Lys Arg Thr Val Glu Thr Ser Ser Phe Tyr Gln Gly Trp195 200 205Leu Asp Asn Val Glu Lys Asp Leu Ser Gln Val His Glu Ala Ile Lys210 215 220Thr Lys Asp Phe Pro Arg Leu Gly Glu Ile Ile Glu Ala Asn Gly Leu225 230 235 240Arg Met His Gly Thr Thr Leu Gly Ala Val Pro Pro Phe Thr Tyr Trp245 250 255Ser Pro Gly Ser Leu Gln Ala Met Ala Leu Val Arg Gln Ala Arg Ala260 265 270Lys Gly Ile Pro Cys Tyr Phe Thr Met Asp Ala Gly Pro Asn Val Lys275 280 285Val Leu Val Glu Lys Lys Asn Leu Glu Ala Leu Lys Thr Phe Leu Ser290 295 300Glu His Phe Ser Lys Glu Gln Leu Val Pro Ala Phe Ala Gly Pro Gly305 310 315 320Ile Glu Leu Phe Glu Thr Lys Gly Met Asp Lys325 330<210>88<211>325<212>PRT<213>屎肠球菌(Enterococcus faecium)<400>88Met Phe Lys Gly Lys Ala Arg Ala Tyr Thr Asn Ile Ala Leu Ile Lys1 5 10 15Tyr Trp Gly Lys Lys Asn Glu Glu Leu Ile Leu Pro Met Asn Asn Ser20 25 30Leu Ser Leu Thr Leu Asp Ala Phe Tyr Thr Glu Thr Glu Val Ile Phe35 40 45Ser Asp Ser Tyr Met Val Asp Glu Phe Tyr Leu Asp Gly Thr Leu Gln50 55 60Asp Glu Lys Ala Thr Lys Lys Val Ser Gln Phe Leu Asp Leu Phe Arg65 70 75 80Lys Glu Ala Gly Leu Ser Leu Lys Ala Ser Val Ile Ser Gln Asn Phe
85 90 95Val Pro Thr Ala Ala Gly Leu Ala Ser Ser Ala Ser Gly Leu Ala Ala100 105 110Leu Ala Gly Ala Cys Asn Thr Ala Leu Lys Leu Gly Leu Asp Asp Leu115 120 125Ser Leu Ser Arg Phe Ala Arg Arg Gly Ser Gly Ser Ala Cys Arg Ser130 135 140Ile Phe Gly Gly Phe Val Glu Trp Glu Lys Gly His Asp Asp Leu Ser145 150 155 160Ser Tyr Ala Lys Pro Val Pro Ser Asp Ser Phe Glu Asp Asp Leu Ala165 170 175Met Val Phe Val Leu Ile Asn Asp Gln Lys Lys Glu Val Ser Ser Arg180 185 190Asn Gly Met Arg Arg Thr Val Glu Thr Ser Asn Phe Tyr Gln Gly Trp195 200 205Leu Asp Ser Val Glu Gly Asp Leu Tyr Gln Leu Lys Gln Ala Ile Lys210 215 220Thr Lys Asp Phe Gln Leu Leu Gly Glu Thr Met Glu Arg Asn Gly Leu225 230 235 240Lys Met His Gly Thr Thr Leu Ala Ala Gln Pro Pro Phe Thr Tyr Trp245 250 255Ser Pro Asn Ser Leu Lys Ala Met Asp Ala Val Arg Gln Leu Arg Lys260 265 270Gln Gly Ile Pro Cys Tyr Phe Thr Met Asp Ala Gly Pro Asn Val Lys275 280 285Val Leu Val Glu Asn Ser His Leu Ser Glu Val Gln Glu Thr Phe Thr290 295 300Lys Leu Phe Ser Lys Glu Gln Val Ile Thr Ala His Ala Gly Pro Gly305 310 315 320Ile Ala Ile Ile Glu325<210>89<211>327<212>PRT<213>溶血葡萄球菌(Staphylococcus haemolyticus)<400>89Met Lys Lys Ser Gly Lys Ala Arg Ala His Thr Asn Ile Ala Leu Ile1 5 10 15Lys Tyr Trp Gly Lys Ala Asp Glu Ala Leu Ile Ile Pro Met Asn Asn20 25 30Ser Leu Ser Val Thr Leu Asp Arg Phe Tyr Thr Glu Thr Arg Val Thr35 40 45
Phe Asp Glu Thr Leu Thr Glu Asp Gln Leu Ile Leu Asn Gly Glu Ala50 55 60Val Asn Ala Lys Glu Ser Ala Lys Ile Gln Arg Tyr Met Glu Met Ile65 70 75 80Arg Lys Glu Ala Gly Ile Ser His Glu Ala Leu Ile Glu Ser Glu Asn85 90 95Phe Val Pro Thr Ala Ala Gly Leu Ala Ser Ser Ala Ser Ala Tyr Ala100 105 110Ala Leu Ala Gly Ala Cys Asn Glu Ala Leu Gln Leu Gly Leu Ser Asp115 120 125Lys Asp Leu Ser Arg Leu Ala Arg Arg Gly Ser Gly Ser Ala Ser Arg130 135 140Ser Ile Tyr Gly Gly Phe Ala Glu Trp Glu Lys Gly Asn Asp Asp Glu145 150 155 160Thr Ser Phe Ala His Arg Val Glu Ala Asp Gly Trp Glu Asn Glu Leu165 170 175Ala Met Val Phe Val Val Ile Asn Asn Lys Ser Lys Lys Val Ser Ser180 185 190Arg Ser Gly Met Ser Leu Thr Arg Asp Thr Ser Arg Phe Tyr Gln Tyr195 200 205Trp Leu Asp Asn Val Glu Pro Asp Leu Lys Glu Thr Lys Glu Ala Ile210 215 220Ala Gln Lys Asp Phe Lys Arg Met Gly Glu Val Ile Glu Ala Asn Gly225 230 235 240Leu Arg Met His Ala Thr Asn Leu Gly Ala Gln Pro Pro Phe Thr Tyr245 250 255Leu Val Pro Glu Ser Tyr Asp Ala Met Arg Ile Val His Glu Cys Arg260 265 270Glu Ala Gly Leu Pro Cys Tyr Phe Thr Met Asp Ala Gly Pro Asn Val275 280 285Lys Val Leu Ile Glu Lys Lys Asn Gln Gln Ala Ile Val Asp Lys Phe290 295 300Leu Gln Glu Phe Asp Gln Ser Gln Ile Ile Thr Ser Asp Ile Thr Gln305 310 315 320Ser Gly Val Glu Ile Ile Lys325<210>90<211>327<212>PRT<213>表皮葡萄球菌(Staphylococcus epidermidis)<400>90Met Val Lys Ser Gly Lys Ala Arg Ala His Thr Asn Ile Ala Leu Ile
1 5 10 15Lys Tyr Trp Gly Lys Ala Asp Glu Thr Tyr Ile Ile Pro Met Asn Asn20 25 30Ser Leu Ser Val Thr Leu Asp Arg Phe Tyr Thr Glu Thr Lys Val Thr35 40 45Phe Asp Pro Asp Phe Thr Glu Asp Cys Leu Ile Leu Asn Gly Asn Glu50 55 60Val Asn Ala Lys Glu Lys Glu Lys Ile Gln Asn Tyr Met Asn Ile Val65 70 75 80Arg Asp Leu Ala Gly Asn Arg Leu His Ala Arg Ile Glu Ser Glu Asn85 90 95Tyr Val Pro Thr Ala Ala Gly Leu Ala Ser Ser Ala Ser Ala Tyr Ala100 105 110Ala Leu Ala Ala Ala Cys Asn Glu Ala Leu Ser Leu Asn Leu Ser Asp115 120 125Thr Asp Leu Ser Arg Leu Ala Arg Arg Gly Ser Gly Ser Ala Ser Arg130 135 140Ser Ile Phe Gly Gly Phe Ala Glu Trp Glu Lys Gly His Asp Asp Leu145 150 155 160Thr Ser Tyr Ala His Gly Ile Asn Ser Asn Gly Trp Glu Lys Asp Leu165 170 175Ser Met Ile Phe Val Val Ile Asn Asn Gln Ser Lys Lys Val Ser Ser180 185 190Arg Ser Gly Met Ser Leu Thr Arg Asp Thr Ser Arg Phe Tyr Gln Tyr195 200 205Trp Leu Asp His Val Asp Glu Asp Leu Asn Glu Ala Lys Glu Ala Val210 215 220Lys Asn Gln Asp Phe Gln Arg Leu Gly Glu Val Ile Glu Ala Asn Gly225 230 235 240Leu Arg Met His Ala Thr Asn Leu Gly Ala Gln Pro Pro Phe Thr Tyr245 250 255Leu Val Gln Glu Ser Tyr Asp Ala Met Ala Ile Val Glu Gln Cys Arg260 265 270Lys Ala Asn Leu Pro Cys Tyr Phe Thr Met Asp Ala Gly Pro Asn Val275 280 285Lys Val Leu Val Glu Lys Lys Asn Lys Gln Ala Val Met Glu Gln Phe290 295 300Leu Lys Val Phe Asp Glu Ser Lys Ile Ile Ala Ser Asp Ile Ile Ser305 310 315 320Ser Gly Val Glu Ile Ile Lys325
<210>91<211>327<212>PRT<213>金黄色葡萄球菌(Staphylococcus aureus)<400>91Met Ile Lys Ser Gly Lys Ala Arg Ala His Thr Asn Ile Ala Leu Ile1 5 10 15Lys Tyr Trp Gly Lys Lys Asp Glu Ala Leu Ile Ile Pro Met Asn Asn20 25 30Ser Ile Ser Val Thr Leu Glu Lys Phe Tyr Thr Glu Thr Lys Val Thr35 40 45Phe Asn Asp Gln Leu Thr Gln Asp Gln Phe Trp Leu Asn Gly Glu Lys50 55 60Val Ser Gly Lys Glu Leu Glu Lys Ile Ser Lys Tyr Met Asp Ile Val65 70 75 80Arg Asn Arg Ala Gly Ile Asp Trp Tyr Ala Glu Ile Glu Ser Asp Asn85 90 95Phe Val Pro Thr Ala Ala Gly Leu Ala Ser Ser Ala Ser Ala Tyr Ala100 105 110Ala Leu Ala Ala Ala Cys Asn Gln Ala Leu Asp Leu Gln Leu Ser Asp115 120 125Lys Asp Leu Ser Arg Leu Ala Arg Ile Gly Ser Gly Ser Ala Ser Arg130 135 140Ser Ile Tyr Gly Gly Phe Ala Glu Trp Glu Lys Gly Tyr Asn Asp Glu145 150 155 160Thr Ser Tyr Ala Val Pro Leu Glu Ser Asn His Phe Glu Asp Asp Leu165 170 175Ala Met Ile Phe Val Val Ile Asn Gln His Ser Lys Lys Val Pro Ser180 185 190Arg Tyr Gly Met Ser Leu Thr Arg Asn Thr Ser Arg Phe Tyr Gln Tyr195 200 205Trp Leu Asp His Ile Asp Glu Asp Leu Ala Glu Ala Lys Ala Ala Ile210 215 220Gln Asp Lys Asp Phe Lys Arg Leu Gly Glu Val Ile Glu Glu Asn Gly225 230 235 240Leu Arg Met His Ala Thr Asn Leu Gly Ser Thr Pro Pro Phe Thr Tyr245 250 255Leu Val Gln Glu Ser Tyr Asp Val Met Ala Leu Val His Glu Cys Arg260 265 270Glu Ala Gly Tyr Pro Cys Tyr Phe Thr Met Asp Ala Gly Pro Asn Val275 280 285
Lys Ile Leu Val Glu Lys Lys Asn Lys Gln Gln Ile Ile Asp Lys Leu290 295 300Leu Thr Gln Phe Asp Asn Asn Gln Ile Ile Asp Ser Asp Ile Ile Ala305 310 315 320Thr Gly Ile Glu Ile Ile Glu325<210>92<211>350<212>PRT<213>链霉菌属菌种(Streptomyces sp.)<400>92Met Arg Ser Glu His Pro Thr Thr Thr Val Leu Gln Ser Arg Glu Gln1 5 10 15Gly Ser Ala Ala Gly Ala Thr Ala Val Ala His Pro Asn Ile Ala Leu20 25 30Ile Lys Tyr Trp Gly Lys Arg Asp Glu Arg Leu Ile Leu Pro Cys Thr35 40 45Thr Ser Leu Ser Met Thr Leu Asp Val Phe Pro Thr Thr Thr Glu Val50 55 60Arg Leu Asp Pro Ala Ala Glu His Asp Thr Ala Ala Leu Asn Gly Glu65 70 75 80Val Ala Thr Gly Glu Thr Leu Arg Arg Ile Ser Ala Phe Leu Ser Leu85 90 95Val Arg Glu Val Ala Gly Ser Asp Gln Arg Ala Val Val Asp Thr Arg100 105 110Asn Thr Val Pro Thr Gly Ala Gly Leu Ala Ser Ser Ala Ser Gly Phe115 120 125Ala Ala Leu Ala Val Ala Ala Ala Ala Ala Tyr Gly Leu Glu Leu Asp130 135 140Asp Arg Gly Leu Ser Arg Leu Ala Arg Arg Gly Ser Gly Ser Ala Ser145 150 155 160Arg Ser Ile Phe Gly Gly Phe Ala Val Trp His Ala Gly Pro Asp Gly165 170 175Thr Ala Thr Glu Ala Asp Leu Gly Ser Tyr Ala Glu Pro Val Pro Ala180 185 190Ala Asp Leu Asp Pro Ala Leu Val Ile Ala Val Val Asn Ala Gly Pro195 200 205Lys Pro Val Ser Ser Arg Glu Ala Met Arg Arg Thr Val Asp Thr Ser210 215 220Pro Leu Tyr Arg Pro Trp Ala Asp Ser Ser Lys Asp Asp Leu Asp Glu225 230 235 240Met Arg Ser Ala Leu Leu Arg Gly Asp Leu Glu Ala Val Gly Glu Ile245 250 255
Ala Glu Arg Asn Ala Leu Gly Met His Ala Thr Met Leu Ala Ala Arg260 265 270Pro Ala Val Arg Tyr Leu Ser Pro Ala Thr Val Thr Val Leu Asp Ser275 280 285Val Leu Gln Leu Arg Lys Asp Gly Val Leu Ala Tyr Ala Thr Met Asp290 295 300Ala Gly Pro Asn Val Lys Val Leu Cys Arg Arg Ala Asp Ala Glu Arg305 310 315 320Val Ala Asp Val Val Arg Ala Ala Ala Ser Gly Gly Gln Val Leu Val325 330 335Ala Gly Pro Gly Asp Gly Ala Arg Leu Leu Ser Glu Gly Ala340 345 350<210>93<211>331<212>PRT<213>灰孢链霉菌(Streptomyces griseosporeus)<400> 93Ala Thr Ala Val Ala Gln Pro Asn Ile Ala Leu Ile Lys Tyr Trp Gly1 5 10 15Lys Lys Asp Glu His Leu Val Leu Pro Arg Thr Asp Ser Leu Ser Met20 25 30Thr Leu Asp Ile Phe Pro Thr Thr Thr Arg Val Gln Leu Ala Pro Gly35 40 45Ala Gly Gln Asp Thr Val Ala Phe Asn Gly Glu Pro Ala Thr Gly Glu50 55 60Ala Glu Arg Arg Ile Thr Ala Phe Leu Arg Leu Val Arg Glu Arg Ser65 70 75 80Gly Arg Thr Glu Arg Ala Arg Val Glu Thr Glu Asn Thr Val Pro Thr85 90 95Gly Ala Gly Leu Ala Ser Ser Ala Ser Gly Phe Ala Ala Leu Ala Val100 105 110Ala Ala Ala Ala Ala Tyr Gly Leu Gly Leu Asp Ala Arg Gly Leu Ser115 120 125Arg Leu Ala Arg Arg Gly Ser Gly Ser Ala Ser Arg Ser Ile Phe Asp130 135 140Gly Phe Ala Val Trp His Ala Gly His Ala Gly Gly Thr Pro Glu Glu145 150 155 160Ala Asp Leu Gly Ser Tyr Ala Glu Pro Val Pro Ala Val Asp Leu Glu165 170 175Pro Ala Leu Val Val Ala Val Val Ser Ala Ala Pro Lys Ala Val Ser180 185 190
Ser Arg Glu Ala Met Arg Arg Thr Val Asp Thr Ser Pro Leu Tyr Glu195 200 205Pro Trp Ala Val Ser Ser Arg Ala Asp Leu Ala Asp Ile Gly Ala Ala210 215 220Leu Ala Arg Gly Asn Leu Pro Ala Val Gly Glu Ile Ala Glu Arg Asn225 230 235 240Ala Leu Gly Met His Ala Thr Met Leu Ala Ala Arg Pro Ala Val Arg245 250 255Tyr Leu Ser Pro Ala Ser Leu Ala Val Leu Asp Gly Val Leu Gln Leu260 265 270Arg Arg Asp Gly Val Pro Ala Tyr Ala Thr Met Asp Ala Gly Pro Asn275 280 285Val Lys Val Leu Cys Pro Arg Ser Asp Ala Glu Arg Val Ala Glu Ala290 295 300Leu Arg Ala Ala Ala Pro Val Gly Ala Val His Ile Ala Gly Pro Gly305 310 315 320Arg Gly Ala Arg Leu Val Ala Glu Glu Cys Arg325 330<210>94<211>312<212>PRT<213>布氏疏螺旋体(Borrelia burgdorferi)<400>94Met Lys Ile Lys Cys Lys Val His Ala Ser Leu Ala Leu Ile Lys Tyr1 5 10 15Trp Gly Lys Lys Asp Val Phe Leu Asn Ile Pro Ala Thr Ser Ser Leu20 25 30Ala Val Ser Val Asp Lys Phe Tyr Ser Ile Ser Glu Leu Glu Leu Ser35 40 45Asn Arg Asp Glu Ile Ile Leu Asn Ser Lys Pro Val Ile Leu Lys Asn50 55 60Arg Glu Lys Val Phe Phe Asp Tyr Ala Arg Lys Ile Leu Asn Glu Pro65 70 75 80Asn Val Arg phe Lys Ile Lys Ser Lys Asn Asn Phe Pro Thr Ala Ala85 90 95Gly Leu Ala Ser Ser Ser Ser Gly Phe Ala Ser Ile Ala Ala Cys Ile100 105 110Leu Lys Tyr Phe Asn Lys Tyr Ser Cys Asn Ser Ala Ser Asn Leu Ala115 120 125Arg Val Gly Ser Ala Ser Ala Ala Arg Ala Ile Tyr Gly Gly Phe Thr130 135 140Ile Leu Lys Glu Gly Ser Lys Glu Ser Phe Gln Leu Arg Asp Gln Ser145 150 155 160
Tyr Phe Asn Asp Leu Arg Ile Ile Phe Ala Ile Ile Asp Ser Asn Glu165 170 175Lys Glu Leu Ser Ser Arg Ala Ala Met Asn Ile Cys Lys Arg His Lys180 185 190Phe Tyr Tyr Asp Ala Trp Ile Ala Ser Ser Lys Lys Ile Phe Lys Asp195 200 205Ala Leu Tyr Phe Phe Leu Lys Lys Asp Phe Ile His Phe Gly Ala Thr210 215 220Ile Val Lys Ser Tyr Gln Asn Met Phe Ala Leu Met Phe Ala Ser Ser225 230 235 240Ile Phe Tyr Phe Lys Asn Ser Thr Ile Asp Leu Ile Arg Tyr Ala Ala245 250 255Asp Leu Arg Asn Glu Gly Ile Phe Val Phe Glu Thr Met Asp Ala Gly260 265 270Pro Gln Val Lys phe Leu Cys Leu Glu Glu Asn Leu Asn Thr Ile Leu275 280 285Lys Gly Leu Lys Gln Asn Phe Thr Gly Ile Asp Phe Ile Val Ser Lys290 295 300Val Gly Cys Asp Leu Glu Trp Ile305 310<210>95<211>292<212>PRT<213>肺炎链球菌(Streptococcus pneumoniae)<400>95Met Thr Lys Lys Val Gly Val Gly Gln Ala His Ser Lys Ile Ile Leu1 5 10 15Ile Gly Glu His Ala Val Val Tyr Gly Tyr Pro Ala Ile Ser Leu Pro20 25 30Leu Leu Glu Val Glu Val Thr Cys Lys Val Val Ser Ala Glu Ser Pro35 40 45Trp Arg Leu Tyr Glu Glu Asp Thr Leu Ser Met Ala Val Tyr Ala Ser50 55 60Leu Glu Tyr Leu Asp Ile Thr Glu Ala Cys Val Arg Cys Glu Ile Asp65 70 75 80Ser Ala Ile Pro Glu Lys Arg Gly Met Gly Ser Ser Ala Ala Ile Ser85 90 95Ile Ala Ala Ile Arg Ala Val Phe Asp Tyr Tyr Gln Ala Asp Leu Pro100 105 110His Asp Val Leu Glu Ile Leu Val Asn Arg Ala Glu Met Ile Ala His115 120 125
Met Asn Pro Ser Gly Leu Asp Ala Lys Thr Cys Leu Ser Asp Gln Pro130 135 140Ile Arg Phe Ile Lys Asn Val Gly Phe Thr Glu Leu Glu Met Asp Leu145 150 155 160Ser Ala Tyr Leu Val Ile Ala Asp Thr Gly Val Tyr Gly His Thr Arg165 170 175Glu Ala Ile Gln Val Val Gln Asn Lys Gly Lys Asp Ala Leu Pro Phe180 185 190Leu His Ala Leu Gly Glu Leu Thr Gln Gln Ala Glu Val Ala Ile Ser195 200 205Gln Lys Tyr Ala Glu Gly Leu Gly Leu Ile Phe Ser Gln Ala His Leu210 215 220His Leu Lys Glu Ile Gly Val Ser Ser Pro Glu Ala Asp Phe Leu Val225 230 235 240Glu Thr Ala Leu Ser Tyr Gly Ala Leu Gly Ala Lys Met Ser Gly Gly245 250 255Gly Leu Gly Gly Cys Ile Ile Ala Leu Val Thr Asn Leu Thr His Ala260 265 270Gln Glu Leu Ala Glu Arg Leu Glu Glu Lys Gly Ala Val Gln Thr Trp275 280 285Ile Glu Ser Leu290<210>96<211>292<212>PRT<213>酿脓链球菌(Streptococcus pyogenes)<400>96Met Asn Glu Asn Ile Gly Tyr Gly Lys Ala His Ser Lys Ile Ile Leu1 5 10 15Ile Gly Glu His Ala Val Val Tyr Gly Tyr Pro Ala Ile Ala Leu Pro20 25 30Leu Thr Asp Ile Glu Val Val Cys His Ile Phe Pro Ala Asp Lys Pro35 40 45Leu Val Phe Asp Phe Tyr Asp Thr Leu Ser Thr Ala Ile Tyr Ala Ala50 55 60Leu Asp Tyr Leu Gln Arg Leu Gln Glu Pro Ile Ala Tyr Glu Ile Val65 70 75 80Ser Gln Val Pro Gln Lys Arg Gly Met Gly Ser Ser Ala Ala Val Ser85 90 95Ile Ala Ala Ile Arg Ala Val Phe Ser Tyr Cys Gln Glu Pro Leu Ser100 105 110Asp Asp Leu Leu Glu Ile Leu Val Asn Lys Ala Glu Ile Ile Ala His115 120 125
Thr Asn Pro Ser Gly Leu Asp Ala Lys Thr Cys Leu Ser Asp His Ala130 135 140Ile Lys Phe Ile Arg Asn Ile Gly Phe Glu Thr Ile Glu Ile Ala Leu145 150 155 160Asn Gly Tyr Leu Ile Ile Ala Asp Thr Gly Ile His Gly His Thr Arg165 170 175Glu Ala Val Asn Lys Val Ala Gln Phe Glu Glu Thr Asn Leu Pro Tyr180 185 190Leu Ala Lys Leu Gly Ala Leu Thr Gln Ala Leu Glu Arg Ala Ile Asn195 200 205Gln Lys Asn Lys Val Ala Ile Gly Gln Leu Met Thr Gln Ala His Ser210 215 220Ala Leu Lys Ala Ile Gly Val Ser Ile Ser Lys Ala Asp Gln Leu Val225 230 235 240Glu Ala Ala Leu Arg Ala Gly Ala Leu Gly Ala Lys Met Thr Gly Gly245 250 255Gly Leu Gly Gly Cys Met Ile Ala Leu Ala Asp Thr Lys Asp Met Ala260 265 270Glu Lys Ile Ser His Lys Leu Lys Glu Glu Gly Ala Val Asn Thr Trp275 280 285Ile Gln Met Leu290<210>97<211>314<212>PRT<213>粪肠球菌(Enterococcus faecalis)<400>97Met Asn Ile Lys Lys Gln Gly Leu Gly Gln Ala Thr Gly Lys Ile Ile1 5 10 15Leu Met Gly Glu His Ala Val Val Tyr Gly Glu Pro Ala Ile Ala Phe20 25 30Pro Phe Gln Ala Thr Glu Ile Thr Ala Val Phe Thr Pro Ala Lys Thr35 40 45Met Gln Ile Asp Cys Ala Tyr Phe Thr Gly Leu Leu Glu Asp Val Pro50 55 60Gln Glu Leu Ala Asn Ile Lys Glu Val Val Gln Gln Thr Leu His Phe65 70 75 80Leu Lys Glu Asp Thr Phe Lys Gly Thr Leu Thr Leu Thr Ser Thr Ile85 90 95Pro Ala Glu Arg Gly Met Gly Ser Ser Ala Ala Thr Ala Val Ala Ile100 105 110Val Arg Ser Leu Phe Asp Tyr Phe Asp Tyr Ala Tyr Thr Tyr Gln Glu
115 120 125Leu Phe Glu Leu Val Ser Leu Ser Glu Lys Ile Ala His Gly Asn Pro130 135 140Ser Gly Ile Asp Ala Ala Ala Thr Ser Gly Ala Asp Pro Leu Phe Phe145 150 155 160Thr Arg Gly Phe Pro Pro Thr His Phe Ser Met Asn Leu Ser Asn Ala165 170 175Tyr Leu Val Val Ala Asp Thr Gly Ile Lys Gly Gln Thr Arg Glu Ala180 185 190Val Lys Asp Ile Ala Gln Leu Ala Gln Asn Asn Pro Thr Ala Ile Ala195 200 205Glu Thr Met Lys Gln Leu Gly Ser Phe Thr Lys Glu Ala Lys Gln Ala210 215 220Ile Leu Gln Asp Asp Lys Gln Lys Leu Gly Gln Leu Met Thr Leu Ala225 230 235 240Gln Glu Gln Leu Gln Gln Leu Thr Val Ser Asn Asp Met Leu Asp Arg245 250 255Leu Val Ala Leu Ser Leu Glu His Gly Ala Leu Gly Ala Lys Leu Thr260 265 270Gly Gly Gly Arg Gly Gly Cys Met Ile Ala Leu Thr Asp Asn Lys Lys275 280 285Thr Ala Gln Thr Ile Ala Gln Thr Leu Glu Glu Asn Gly Ala Val Ala290 295 300Thr Trp Ile Gln Ser Leu Glu Val Lys Lys305 310<210>98<211>314<212>PRT<213>屎肠球菌(Enterococcus faecium)<400>98Met Ala Asn Tyr Gly Gln Gly Glu Ser Ser Gly Lys Ile Ile Leu Met1 5 10 15Gly Glu His Ala Val Val Tyr Gly Glu Pro Ala Ile Ala Phe Pro Phe20 25 30Tyr Ala Thr Lys Val Thr Ala Phe Leu Glu Glu Leu Asp Ala Met Asp35 40 45Asp Gln Leu Val Ser Ser Tyr Tyr Ser Gly Asn Leu Ala Glu Ala Pro50 55 60His Ala Leu Lys Asn Ile Lys Lys Leu Phe Ile His Lau Lys Lys Gln65 70 75 80His Asp Ile Gln Lys Asn Leu Gln Leu Thr Ile Glu Ser Thr Ile Pro85 90 95
Ala Glu Arg Gly Met Gly Ser Ser Ala Ala Val Ala Thr Ala Val Thr100 105 110Arg Ala Phe Tyr Asp Tyr Leu Ala Phe Pro Leu Ser Arg Glu Ile Leu115 120 125Leu Glu Asn Val Gln Leu Ser Glu Lys Ile Ala His Gly Asn Pro Ser130 135 140Gly Ile Asp Ala Ala Ala Thr Ser Ser Leu Gln Pro Ile Tyr Phe Thr145 150 155 160Lys Gly His Pro Phe Asp Tyr Phe Ser Leu Asn Ile Asp Ala Phe Leu165 170 175Ile Val Ala Asp Thr Gly Ile Lys Gly Gln Thr Arg Glu Ala Val Lys180 185 190Asp Val Ala His Leu Phe Glu Thr Gln Pro His Glu Thr Gly Gln Met195 200 205Ile Gln Lys Leu Gly Tyr Leu Thr Lys Gln Ala Lys Gln Ala Ile Ile210 215 220Glu Asn Ser Pro Glu Thr Leu Ala Gln Thr Met Asp Glu Ser Gln Ser225 230 235 240Leu Leu Glu Lys Leu Thr Ile Ser Asn Asp Phe Leu Asn Leu Leu Ile245 250 255Gln Thr Ala Lys Asp Thr Gly Ala Leu Gly Ala Lys Leu Thr Gly Gly260 265 270Gly Arg Gly Gly Cys Met Ile Ala Leu Ala Gln Thr Lys Thr Lys Ala275 280 285Gln Glu Ile Ser Gln Ala Leu Glu Asp Ala Gly Ala Ala Glu Thr Trp290 295 300Ile Gln Gly Leu Gly Val His Thr Tyr Val305 310<210>99<211>307<212>PRT<213>溶血葡萄球菌(Staphylococcus haemolyticus)<400>99Met Val Gln Arg Gly Tyr Gly Glu Ser Asn Gly Lys Ile Ile Leu Ile1 5 10 15Gly Glu His Ala Val Thr Phe Gly Glu Pro Ala Ile Ala Ile Pro Phe20 25 30Thr Ser Gly Lys Val Lys Val Leu Ile Glu Ser Leu Glu Lys Gly Asn35 40 45Tyr Ser Ala Ile Gln Ser Asp Val Tyr Asp Gly Pro Leu Tyr Asp Ala50 55 60Pro Glu His Leu Lys Ser Leu Ile Gly His Phe Val Glu Asn Lys Lys65 70 75 80
Val Glu Glu Pro Leu Leu Ile Lys Ile Gln Ala Asn Leu Pro Pro Ser85 90 95Arg Gly Leu Gly Ser Ser Ala Ala Val Ala Val Ala Phe Ile Arg Ala100 105 110Ser Tyr Asp Tyr Leu Gly Leu Pro Leu Thr Asp Lys Glu Leu Leu Glu115 120 125Asn Ala Asp Trp Ala Glu Arg Ile Ala His Gly Lys Pro Ser Gly Ile130 135 140Asp Thr Lys Thr Ile Val Thr Asn Gln Pro Val Trp Tyr Gln Lys Gly145 150 155 160Glu Val Glu Ile Leu Lys Thr Leu Asp Leu Asp Gly Tyr Met Val Val165 170 175Ile Asp Thr Gly Val Lys Gly Ser Thr Lys Gln Ala Val Glu Asp Val180 185 190His Gln Leu Cys Asp Asn Asp Lys Asn Tyr Met Gln Val Val Lys His195 200 205Ile Gly Ser Leu Val Tyr Ser Ala Ser Glu Ala Ile Glu His His Ser210 215 220Phe Asp Gln Leu Ala Thr Ile Phe Asn Gln Cys Gln Asp Asp Leu Arg225 230 235 240Thr Leu Thr Val Ser His Asp Lys Ile Glu Met Phe Leu Arg Leu Gly245 250 255Glu Glu Asn Gly Ser Val Ala Gly Lys Leu Thr Gly Gly Gly Arg Gly260 265 270Gly Ser Met Leu Ile Leu Ala Lys Glu Leu Gln Thr Ala Lys Asn Ile275 280 285Val Ala Ala Val Glu Lys Ala Gly Ala Gln His Thr Trp Ile Glu Lys290 295 300Leu Gly Gly305<210>100<211>306<212>PRT<213>表皮葡萄球菌(Staphylococcus epidermidis)<400>100Met Thr Arg Gln Gly Tyr Gly Glu Ser Thr Gly Lys Ile Ile Leu Met1 5 10 15Gly Glu His Ala Val Thr Phe Gly Gln Pro Ala Ile Ala Ile Pro Phe20 25 30Asn Ala Gly Lys Ile Lys Val Leu Ile Glu Ser Leu Asp Glu Gly Asn35 40 45Tyr Ser Ser Ile Thr Ser Asp Val Tyr Asp Gly Met Leu Tyr Asp Ala
50 55 60Pro Glu His Leu Lys Ser Ile Ile Asn Arg Phe Val Glu Lys Ser Gly65 70 75 80Val Lys Glu Pro Leu Ser Val Lys Ile Gln Thr Asn Leu Pro Pro Ser85 90 95Arg Gly Leu Gly Ser Ser Ala Ala Val Ala Val Ala Phe Val Arg Ala100 105 110Ser Tyr Asp Phe Met Asp Gln Pro Leu Asp Asp Lys Thr Leu Ile Lys115 120 125Glu Ala Asn Trp Ala Glu Gln Ile Ala His Gly Lys Pro Ser Gly Ile130 135 140Asp Thr Gln Thr Ile Val Ser Asn Lys Pro Val Trp Phe Lys Gln Gly145 150 155 160Gln Ala Glu Thr Leu Lys Ser Leu Lys Leu Asn Gly Tyr Met Val Val165 170 175Ile Asp Thr Gly Val Lys Gly Ser Thr Lys Gln Ala Val Glu Asp Val180 185 190His Val Leu Cys Glu Ser Asp Glu Tyr Met Lys Tyr Ile Glu His Ile195 200 205Gly Thr Leu Val His Ser Ala Ser Glu Ser Ile Glu Gln His Asp Phe210 215 220His His Leu Ala Asp Ile Phe Asn Ala Cys Gln Glu Asp Leu Arg His225 230 235 240Leu Thr Val Ser His Asp Lys Ile Glu Lys Leu Leu Gln Ile Gly Lys245 250 255Glu His Gly Ala Ile Ala Gly Lys Leu Thr Gly Gly Gly Arg Gly Gly260 265 270Ser Met Leu Leu Leu Ala Glu Asn Leu Lys Thr Ala Lys Thr Ile Val275 280 285Ala Ala Val Glu Lys Ala Gly Ala Ala His Thr Trp Ile Glu His Leu290 295 300Gly Gly305<210>101<211>306<212>PRT<213>金黄色葡萄球菌(Staphylococcus aureus)<400>101Met Thr Arg Lys Gly Tyr Gly Glu Ser Thr Gly Lys Ile Ile Leu Ile1 5 10 15GlV Glu His Ala Val Thr Phe Gly Glu Pro Ala Ile Ala Val Pro Phe20 25 30
Asn Ala Gly Lys Ile Lys Val Leu Ile Glu Ala Leu Glu Ser Gly Asn35 40 45Tyr Ser Ser Ile Lys Ser Asp Val Tyr Asp Gly Met Leu Tyr Asp Ala50 55 60Pro Asp His Leu Lys Ser Leu Val Asn Arg Phe Val Glu Leu Asn Asn65 70 75 80Ile Thr Glu Pro Leu Ala Val Thr Ile Gln Thr Asn Leu Pro Pro Ser85 90 95Arg Gly Leu Gly Ser Ser Ala Ala Val Ala Val Ala Phe Val Arg Ala100 105 110Ser Tyr Asp Phe Leu Gly Lys Ser Leu Thr Lys Glu Glu Leu Ile Glu115 120 125Lys Ala Asn Trp Ala Glu Gln Ile Ala His Gly Lys Pro Ser Gly Ile130 135 140Asp Thr Gln Thr Ile Val Ser Gly Lys Pro Val Trp Phe Gln Lys Gly145 150 155 160Gln Ala Glu Thr Leu Lys Thr Leu Ser Leu Asp Gly Tyr Met Val Val165 170 175Ile Asp Thr Gly Val Lys Gly Ser Thr Arg Gln Ala Val Glu Asp Val180 185 190His Lys Leu Cys Glu Asp Pro Gln Tyr Met Ser His Val Lys His Ile195 200 205Gly Lys Leu Val Leu Arg Ala Ser Asp Val Ile Glu His His Asn Phe210 215 220Glu Ala Leu Ala Asp Ile Phe Asn Glu Cys His Ala Asp Leu Lys Ala225 230 235 240Leu Thr Val Ser His Asp Lys Ile Glu Gln Leu Met Lys Ile Gly Lys245 250 255Glu Asn Gly Ala Ile Ala Gly Lys Leu Thr Gly Ala Gly Arg Gly Gly260 265 270Ser Met Leu Leu Leu Ala Lys Asp Leu Pro Thr Ala Lys Asn Ile Val275 280 285Lys Ala Val Glu Lys Ala Gly Ala Ala His Thr Trp Ile Glu Asn Leu290 295 300Gly Gly305<210>102<211>345<212>PRT<213>链霉菌属菌种(Streptomyces sp.)<400>102Met Gln Lys Arg Gln Arg Glu Leu Ser Ala Leu Thr Leu Pro Thr Ser1 5 10 15
Ala Glu Gly Val Ser Glu Ser His Arg Ala Arg Ser Val Gly Ile Gly20 25 30Arg Ala His Ala Lys Ala Ile Leu Leu Gly Glu His Ala Val Val Tyr35 40 45Gly Ala Pro Ala Leu Ala Leu Pro Ile Pro Gln Leu Thr Val Thr Ala50 55 60Ser Val Gly Trp Ser Ser Glu Ala Ser Asp Ser Ala Gly Gly Leu Ser65 70 75 80Tyr Thr Met Thr Gly Thr Pro Ser Arg Ala Leu Val Thr Gln Ala Ser85 90 95Asp Gly Leu His Arg Leu Thr Ala Glu Phe Met Ala Arg Met Gly Val100 105 110Thr Asn Ala Pro His Leu Asp Val Ile Leu Asp Gly Ala Ile Pro His115 120 125Gly Arg Gly Leu Gly Ser Ser Ala Ala Gly Ser Arg Ala Ile Ala Leu130 135 140Ala Leu Ala Asp Leu Phe Gly His Glu Leu Ala Glu His Thr Ala Tyr145 150155 160Glu Leu Val Gln Thr Ala Glu Asn Met Ala His Gly Arg Ala Ser Gly165 170 175Val Asp Ala Met Thr Val Gly Ala Ser Arg Pro Leu Leu Phe Gln Gln180 185 190Gly Arg Thr Glu Arg Leu Ala Ile Gly Cys Asp Ser Leu Phe Ile Val195 200 205Ala Asp Ser Gly Val Pro Gly Ser Thr Lys Glu Ala Val Glu Met Leu210 215 220Arg Glu Gly Phe Thr Arg Ser Ala Gly Thr Gln Glu Arg Phe Val Gly225 230 235 240Arg Ala Thr Glu Leu Thr Glu Ala Ala Arg Gln Ala Leu Ala Asp Gly245 250 255Arg Pro Glu Glu Leu Gly Ser Gln Leu Thr Tyr Tyr His Glu Leu Leu260 265 270His Glu Ala Arg Leu Ser Thr Asp Gly Ile Asp Ala Leu Val Glu Ala275 280 285Ala Leu Lys Ala Gly Ser Leu Gly Ala Lys Ile Thr Gly Gly Gly Leu290 295 300Gly Gly Cys Met Ile Ala Gln Ala Arg Pro Glu Gln Ala Arg Glu Val305 310 315 320Thr Arg Gln Leu His Glu Ala Gly Ala Val Gln Thr Trp Val Val Pro325 330 335
Leu Lys Gly Leu Asp Asn His Ala Gln340 345<210>103<211>334<212>PRT<213>灰孢链霉菌(Streptomyces griseosporeus)<400>103Met Thr Leu Pro Thr Ser Val Glu Glu Gly Ser Lys Ala His Arg Ala1 5 10 15Arg Ala Val Gly Thr Gly Arg Ala His Ala Lys Ala Ile Leu Leu Gly20 25 30Glu His Ala Val Val Tyr Gly Thr Pro Ala Leu Ala Met Pro Ile Pro35 40 45Gln Leu Ala Val Thr Ala Ser Ala Gly Trp Ser Gly Arg Ser Ala Glu50 55 60Ser Arg Gly Gly Pro Thr Phe Thr Met Thr Gly Ser Ala Ser Arg Ala65 70 75 80Val Thr Ala Gln Ala Leu Asp Gly Leu Arg Arg Leu Thr Ala Ser Val85 90 95Lys Ala His Thr Gly Val Thr Asp Gly Gln His Leu Asp Val Ser Leu100 105 110Asp Gly Ala Ile Pro Pro Gly Arg Gly Leu Gly Ser Ser Ala Ala Asn115 120 125Ala Arg Ala Ile Ile Leu Ala Leu Ala Asp Leu Phe Gly Arg Glu Leu130 135 140Thr Glu Gly Glu Val Phe Asp Leu Val Gln Glu Ala Glu Asn Leu Thr145 150 155 160His Gly Arg Ala Ser Gly Val Asp Ala Val Thr Val Gly Ala Thr Ala165 170 175Pro Leu Leu Phe Arg Ala Gly Thr Ala Gln Ala Leu Asp Ile Gly Cys180 185 190Asp Ala Leu Phe Val Val Ala Asp Ser Gly Thr Ala Gly Ser Thr Lys195 200 205Glu Ala Ile Glu Leu Leu Arg Ala Gly Phe Arg Ala Gly Ala Gly Lys210 215 220Glu Glu Arg Phe Met His Arg Ala Ala His Leu Val Asp Asp Ala Arg225 230 235 240Ala Ser Leu Ala Glu Gly Glu Pro Glu Ala Phe Gly Ser Cys Leu Thr245 250 255Glu Tyr His Gly Leu Leu Arg Gly Ala Gly Leu Ser Thr Asp Arg Ile260 265 270Asp Ala Leu Val Asp Ala Ala Leu Gln Ala Asp Ser Leu Gly Ala Lys
275 280 285Ile Thr Gly Gly Gly Leu Gly Gly Cys Val Leu Ala Met Ser Arg Pro290 295 300Glu Arg Ala Glu Glu Val Ala Arg Gln Leu His Ala Ala Gly Ala Val305 310 315 320Arg Thr Trp Ala Val Gln Leu Arg Arg Ser Thr His Glu Arg325 330<210>104<211>296<212>PRT<213>布氏疏螺旋体(Borrelia burgdorferi)<400>104Met Leu Arg Ile Arg Lys Pro Ala Lys Ile Leu Phe Leu Gly Glu His1 5 10 15Ser Ala Val Tyr Gly Phe Pro Val Ile Gly Ala Thr Val Pro Ile Tyr20 25 30Met Asp Leu Ile Tyr Ser Val Ser Lys Asn Trp Lys Tyr Leu Gly Lys35 40 45Pro Ser Thr Arg Leu Asn Ser Leu Ile Ser Phe Ile Val Ser Asn Tyr50 55 60Ser Lys Val Asn Pro Ile Glu Phe Asp Ile Ile Ser Glu Ile Pro Ile65 70 75 80Gly Val Gly Leu Gly Ser Ser Ala Ser Leu Ser Leu Cys Phe Ala Glu85 90 95Tyr Ile Thr Ser His Phe Glu Tyr Lys Asp Cys Asn Lys Ile Leu Leu100 105 110Ala Asn Gln Ile Glu Asn Ile Phe His Gly Lys Ser Ser Gly Met Asp115 120 125Ile Arg Leu Ile Asp Leu Asn Gly Thr Phe Tyr Leu Glu Lys Lys Glu130 135 140Asn Val Leu His Ser Lys Lys Ile Lys Asp Ser Gly Phe Tyr Phe Leu145 150 155 160Ile Gly Ala Ile Lys Arg Asp Leu Thr Thr Lys Glu Ile Val Val Asn165 170 175Leu Lys Lys Asp Leu Leu Ser Asn Ala Tyr Leu Phe Val Phe Ile Glu180 185 190Lys Leu Gly Leu Ala Val Ser Asn Ser Tyr Ala Ser Phe Gln Asn Lys195 200 205Asp Val Tyr Ser Leu Ala Asn Glu Met Asn Ile Ala Gln Cys Cys Leu210 215 220Lys Arg Leu Gly Leu Ser Asn Asp Thr Leu Asp Trp Leu Ile Ser Glu225 230 235 240
Gly Ile Lys Leu Gly Ala Leu Ser Gly Lys Leu Ser Gly Ala Gly Lys245 250 255Gly Gly Ala Phe Ile Phe Leu Phe Glu Ser Leu Ile Lys Ala Asn Ile260 265 270Val Gln Lys Glu Leu Asn Asn Met Leu Asp Ser Lys Ile Asp Leu Leu275 280 285Leu Lys Leu Lys Val Ile Glu Thr290 295<210>105<211>336<212>PRT<213>肺炎链球菌(Streptococcus pneumoniae)<400>105Met Ile Ala Val Lys Thr Cys Gly Lys Leu Tyr Trp Ala Gly Glu Tyr1 5 10 15Ala Ile Leu Glu Pro Gly Gln Leu Ala Leu Ile Lys Asp Ile Pro Ile20 25 30Tyr Met Arg Ala Glu Ile Ala Phe Ser Asp Ser Tyr Arg Ile Tyr Ser35 40 45Asp Met Phe Asp Phe Ala Val Asp Leu Arg Pro Asn Pro Asp Tyr Ser50 55 60Leu Ile Gln Glu Thr Ile Ala Leu Met Gly Asp Phe Leu Ala Val Arg65 70 75 80Gly Gln Asn Leu Arg Pro Phe Ser Leu Lys Ile Cys Gly Lys Met Glu85 90 95Arg Glu Gly Lys Lys Phe Gly Leu Gly Ser Ser Gly Ser Val Val Val100 105 110Leu Val Val Lys Ala Leu Leu Ala Leu Tyr Asn Leu Ser Val Asp Gln115 120 125Asn Leu Leu Phe Lys Leu Thr Ser Ala Val Leu Leu Lys Arg Gly Asp130 135 140Asn Gly Ser Met Gly Asp Leu Ala Cys Ile Val Ala Glu Asp Leu Val145 150 155 160Leu Tyr Gln Ser Phe Asp Arg Gln Lys Ala Ala Ala Trp Leu Glu Glu165 170 175Glu Asn Leu Ala Thr Val Leu Glu Arg Asp Trp Gly Phe Phe Ile Ser180 185 190Gln Val Lys Pro Thr Leu Glu Cys Asp Phe Leu Val Gly Trp Thr Lys195 200 205Glu Val Ala Val Ser Ser His Met Val Gln Gln Ile Lys Gln Asn Ile210 215 220Asn Gln Asn Phe Leu Ser Ser Ser Lys Glu Thr Val Val Ser Leu Val
225 230 235 240Glu Ala Leu Glu Gln Gly Lys Ala Glu Lys Val Ile Glu Gln Val Glu245 250 255Val Ala Ser Lys Leu Leu Glu Gly Leu Ser Thr Asp Ile Tyr Thr Pro260 265 270Leu Leu Arg Gln Leu Lys Glu Ala Ser Gln Asp Leu Gln Ala Val Ala275 280 285Lys Ser Ser Gly Ala Gly Gly Gly Asp Cys Gly Ile Ala Leu Ser Phe290 295 300Asp Ala Gln Ser Ser Arg Asn Thr Leu Lys Asn Arg Trp Ala Asp Leu305 310 315 320Gly Ile Glu Leu Leu Tyr Gln Glu Arg Ile Gly His Asp Asp Lys Ser325 330 335<210>106<211>335<212>PRT<213>酿脓链球菌(Streptococcus pyogenes)<400>106Met Ser Asn Tyr Cys Val Gln Thr Gly Gly Lys Leu Tyr Leu Thr Gly1 5 10 15Glu Tyr Ala Ile Leu Ile Pro Gly Gln Lys Ala Leu Ile His Phe Ile20 25 30Pro Leu Met Met Thr Ala Glu Ile Ser Pro Ala Ala His Ile Gln Leu35 40 45Ala Ser Asp Met Phe Ser His Lys Ala Gly Met Thr Pro Asp Ala Ser50 55 60Tyr Ala Leu Ile Gln Ala Thr Val Lys Thr Phe Ala Asp Tyr Leu Gly65 70 75 80Gln Ser Ile Asp Gln Leu Glu Pro Phe Ser Leu Ile Ile Thr Gly Lys85 90 95Met Glu Arg Asp Gly Lys Lys Phe Gly Ile Gly Ser Ser Gly Ser Val100 105 110Thr Leu Leu Thr Leu Lys Ala Leu Ser Ala Tyr Tyr Gln Ile Thr Leu115 120 125Thr Pro Glu Leu Leu Phe Lys Leu Ala Ala Tyr Thr Leu Leu Lys Gln130 135 140Gly Asp Asn Gly Ser Met Gly Asp Ile Ala Cys Ile Ala Tyr Gln Thr145 150 155 160Leu Val Ala Tyr Thr Ser Phe Asp Arg Glu Gln Val Ser Asn Trp Leu165 170 175Gln Thr Met Pro Leu Lys Lys Leu Leu Val Lys Asp Trp Gly Tyr His180 185 190
Ile Gln Val Ile Gln Pro Ala Leu Pro Cys Asp Phe Leu Val Gly Trp195 200 205Thr Lys Ile Pro Ala Ile Ser Arg Gln Met Ile Gln Gln Val Thr Ala210 215 220Ser Ile Thr Pro Ala Phe Leu Arg Thr Ser Tyr Gln Leu Thr Gln Ser225 230 235 240Ala Met Val Ala Leu Gln Glu Gly His Lys Glu Glu Leu Lys Lys Ser245 250 255Leu Ala Gly Ala Ser His Leu Leu Lys Glu Leu His Pro Ala Ile Tyr260 265 270His Pro Lys Leu Val Thr Leu Val Ala Ala Cys Gln Lys Gln Asp Ala275 280 285Val Ala Lys Ser Ser Gly Ala Gly Gly Gly Asp Cys Gly Ile Ala Leu290 295 300Ala Phe Asn Gln Asp Ala Arg Asp Thr Leu Ile Ser Lys Trp Gln Glu305 310 315 320Ala Asp Ile Ala Leu Leu Tyr Gln Glu Arg Trp Gly Glu Asn Asp325 330 335<210>107<211>368<212>PRT<213>粪肠球菌(Enterococcus faecalis)<400>107Met Ile Glu Val Thr Thr Pro Gly Lys Leu Phe Ile Ala Gly Glu Tyr1 5 10 15Ala Val Val Glu Pro Gly His Pro Ala Ile Ile Val Ala Val Asp Gln20 25 30Phe Val Thr Val Thr Val Glu Glu Thr Thr Asp Glu Gly Ser Ile Gln35 40 45Ser Ala Gln Tyr Ser Ser Leu Pro Ile Arg Trp Thr Arg Arg Asn Gly50 55 60Glu Leu Val Leu Asp Ile Arg Glu Asn Pro Phe His Tyr Val Leu Ala65 70 75 80Ala Ile His Leu Thr Glu Lys Tyr Ala Gln Glu Gln Asn Lys Glu Leu85 90 95Ser Phe Tyr His Leu Lys Val Thr Ser Glu Leu Asp Ser Ser Asn Gly100 105 110Arg Lys Tyr Gly Leu Gly Ser Ser Gly Ala Val Thr Val Gly Thr Val115 120 125Lys Ala Leu Asn Ile Phe Tyr Asp Leu Gly Leu Glu Asn Glu Glu Ile130 135 140
Phe Lys Leu Ser Ala Leu Ala His Leu Ala Val Gln Gly Asn Gly Ser145 150 155 160Cys Gly Asp Ile Ala Ala Ser Cys Tyr Gly Gly Trp Ile Ala Phe Ser165 170 175Thr Phe Asp His Asp Trp Val Asn Gln Lys Val Thr Thr Glu Thr Leu180 185 190Thr Asp Leu Leu Ala Met Asp Trp Pro Glu Leu Met Ile Phe Pro Leu195 200 205Lys Val Pro Lys Gln Leu Arg Leu Leu Ile Gly Trp Thr Gly Ser Pro210 215 220Ala Ser Thr Ser Asp Leu Val Asp Arg Val His Gln Ser Lys Glu Glu225 230 235 240Lys Gln Ala Ala Tyr Glu Gln Phe Leu Met Lys Ser Arg Leu Cys Val245 250 255Glu Thr Met Ile Asn Gly Phe Asn Thr Gly Lys Ile Ser Val Ile Gln260 265 270Lys Gln Ile Thr Lys Asn Arg Gln Leu Leu Ala Glu Leu Ser Ser Leu275 280 285Thr Gly Val Val Ile Glu Thr Glu Ala Leu Lys Asn Leu Cys Asp Leu290 295 300Ala Glu Ser Tyr Thr Gly Ala Ala Lys Ser Ser Gly Ala Gly Gly Gly305 310 315 320Asp Cys Gly Ile Val Ile Phe Arg Gln Lys Ser Gly Ile Leu Pro Leu325 330 335Met Thr Ala Trp Glu Lys Asp Gly Ile Thr Pro Leu Pro Leu His Val340 345 350Tyr Thr Tyr Gly Gln Lys Glu Cys Lys Glu Lys His Glu Ser Lys Arg355 360 365<210>108<211>361<212>PRT<213>屎肠球菌(Enterococcus faecium)<400>108Met Ile Glu Val Ser Ala Pro Gly Lys Leu Tyr Ile Ala Gly Glu Tyr1 5 10 15Ala Val Val Glu Thr Gly His Pro Ala Val Ile Ala Ala Val Asp Gln20 25 30Phe Val Thr Val Thr Val Glu Ser Ala Arg Lys Val Gly Ser Ile Gln35 40 45Ser Ala Gln Tyr Ser Gly Met Pro Val Arg Trp Thr Arg Arg Asn Gly50 55 60Glu Leu Val Leu Asp Ile Arg Glu Asn Pro Phe His Tyr Ile Leu Ala65 70 75 80
Ala Ile Arg Leu Thr Glu Lys Tyr Ala Gln Glu Lys Asn Ile Leu Leu85 90 95Ser Phe Tyr Asp Leu Lys Val Thr Ser Glu Leu Asp Ser Ser Asn Gly100 105 110Arg Lys Tyr Gly Leu Gly Ser Ser Gly Ala Val Thr Val Ala Thr Val115 120 125Lys Ala Leu Asn Val Phe Tyr Ala Leu Asn Leu Ser Gln Leu Glu Ile130 135 140Phe Lys Ile Ala Ala Leu Ala Asn Leu Ala Val Gln Asp Asn Gly Ser145 150 155 160Cys Gly Asp Ile Ala Ala Ser Cys Tyr Gly Gly Trp Ile Ala Phe Ser165 170 175Thr Phe Asp His Pro Trp Leu Gln Glu Gln Glu Thr Gln His Ser Ile180 185 190Ser Glu Leu Leu Ala Leu Asp Trp Pro Gly Leu Ser Ile Glu Pro Leu195 200 205Ile Ala Pro Glu Asp Leu Arg Leu Leu Ile Gly Trp Thr Gly Ser Pro210 215 220Ala Ser Thr Ser Asp Leu Val Asp Gln Val His Arg Ser Arg Glu Asp225 230 235 240Lys Met Val Ala Tyr Gln Leu Phe Leu Lys Asn Ser Thr Glu Cys Val245 250 255Asn Glu Met Ile Lys Gly Phe Lys Glu Asn Asn Val Thr Leu Ile Gln260 265 270Gln Met Ile Arg Lys Asn Arg Gln Leu Leu His Asp Leu Ser Ala Ile275 280 285Thr Gly Val Val Ile Glu Thr Pro Ala Leu Asn Lys Leu Cys Asn Leu290 295 300Ala Glu Gln Tyr Glu Gly Ala Ala Lys Ser Ser Gly Ala Gly Gly Gly305 310 315 320Asp Cys Gly Ile Val Ile Val Asp Gln Lys Ser Gly Ile Leu Pro Leu325 330 335Met Ser Ala Trp Glu Lys Ala Glu Ile Thr Pro Leu Pro Leu His Val340 345 350Tyr Ser Asp Gln Arg Lys Glu Asn Arg355 360<210>109<211>358<212>PRT<213>溶血葡萄球菌(Staphylococcus haemolyticus)<400>109Met Ile Gln Val Lys Ala Pro Gly Lys Leu Tyr Val Ala Gly Glu Tyr
1 5 10 15Ala Val Thr Glu Pro Gly Tyr Lys Ser Val Leu Ile Ala Val Asp Arg20 25 30Phe Val Thr Ala Ser Ile Glu Ala Ser Asn Ala Val Thr Ser Thr Ile35 40 45His Ser Lys Thr Leu His Tyr Glu Pro Val Thr Phe Asn Arg Asn Glu50 55 60Asp Lys Ile Asp Ile Ser Asp Ala Asn Ala Ala Ser Gln Leu Lys Tyr65 70 75 80Val Val Thr Ala Ile Glu Val Phe Glu Gln Tyr Ala Arg Ser Cys Asn85 90 95Val Lys Leu Lys His Phe His Leu Glu Ile Asp Ser Asn Leu Asp Asp100 105 110Ala Ser Gly Asn Lys Tyr Gly Leu Gly Ser Ser Ala Ala Val Leu Val115 120 125Ser Val Val Lys Ala Leu Asn Glu Phe Tyr Asp Met Gln Leu Ser Asn130 135 140Leu Tyr Ile Tyr Lys Leu Ala Val Ile Ser Asn Met Arg Leu Gln Ser145 150 155 160Leu Ser Ser Cys Gly Asp Ile Ala Val Ser Val Tyr Ser Gly Trp Leu165 170 175Ala Tyr Ser Thr Phe Asp His Asp Trp Val Lys Gln Gln Met Glu Glu180 185 190Thr Ser Val Asn Glu Val Leu Glu Lys Asn Trp Pro Gly Leu His Ile195 200 205Glu Pro Leu Gln Ala Pro Glu Asn Met Glu Val Leu Ile Gly Trp Thr210 215 220Gly Ser Pro Ala Ser Ser Pro His Leu Val Ser Glu Val Lys Arg Leu225 230 235 240Lys Ser Asp Pro Ser Phe Tyr Gly Arg Phe Leu Asp Gln Ser His Thr245 250 255Cys Val Glu Asn Leu Ile Tyr Ala Phe Lys Thr Asp Asn Ile Lys Gly260 265 270Val Gln Lys Met Ile Arg Gln Asn Arg Met Ile Ile Gln Gln Met Asp275 280 285Asn Glu Ala Thr Val Asp Ile Glu Thr Glu Asn Leu Lys Met Leu Cys290 295 300Asp Ile Gly Glu Arg Tyr Gly Ala Ala Ala Lys Thr Ser Gly Ala Gly305 310 315 320Gly Gly Asp Cys Gly Ile Ala Ile Ile Asp Asn Arg Ile Asp Lys Asn
325 330 335Arg Ile Tyr Asn Glu Trp Ala Ser His Gly Ile Lys Pro Leu Lys Phe340 345 350Lys Ile Tyr His Gly Gln355<210>110<211>358<212>PRT<213>表皮葡萄球菌(Staphylococcus epidermidis)<400>110Met Ile Gln Val Lys Ala Pro Gly Lys Leu Tyr Ile Ala Gly Glu Tyr1 5 10 15Ala Val Thr Glu Pro Gly Tyr Lys Ser Ile Leu Ile Ala Val Asn Arg20 25 30Phe Val Thr Ala Thr Ile Glu Ala Ser Asn Lys Val Glu Gly Ser Ile35 40 45His Ser Lys Thr Leu His Tyr Glu Pro Val Lys Phe Asp Arg Asn Glu50 55 60Asp Arg Ile Glu Ile Ser Asp Val Gln Ala Ala Lys Gln Leu Lys Tyr65 70 75 80Val Val Thr Ala Ile Glu Val Phe Glu Gln Tyr Val Arg Ser Cys Asn85 90 95Met Asn Leu Lys His Phe His Leu Thr Ile Asp Ser Asn Leu Ala Asp100 105 110Asn Ser Gly Gln Lys Tyr Gly Leu Gly Ser Ser Ala Ala Val Leu Val115 120 125Ser Val Val Lys Ala Leu Asn Glu Phe Tyr Gly Leu Glu Leu Ser Asn130 135 140Leu Tyr Ile Tyr Lys Leu Ala Val Ile Ala Asn Met Lys Leu Gln Ser145 150 155 160Leu Ser Ser Cys Gly Asp Ile Ala Val Ser Val Tyr Ser Gly Trp Leu165 170 175Ala Tyr Ser Thr Phe Asp His Asp Trp Val Lys Gln Gln Met Glu Glu180 185 190Thr Ser Val Asn Asp Val Leu Glu Lys Asn Trp Pro Gly Leu His Ile195 200 205Glu Pro Leu Gln Ala Pro Glu Asn Met Glu Val Leu Ile Gly Trp Thr210 215 220Gly Ser Pro Ala Ser Ser Pro His Leu Val Ser Glu Val Lys Arg Leu225 230 235 240Lys Ser Asp Pro Ser Phe Tyr Gly Asp Phe Leu Asp Gln Ser His Ala245 250 255
Cys Val Glu Ser Leu Ile Gln Ala Phe Lys Thr Asn Asn Ile Lys Gly260 265 270Val Gln Lys Met Ile Arg Ile Asn Arg Arg Ile Ile Gln Ser Met Asp275 280 285Asn Glu Ala Ser Val Glu Ile Glu Thr Asp Lys Leu Lys Lys Leu Cys290 295 300Asp Val Gly Glu Lys His Gly Gly Ala Ser Lys Thr Ser Gly Ala Gly305 310 315 320Gly Gly Asp Cys Gly Ile Thr Ile Ile Asn Lys Val Ile Asp Lys Asn325 330 335Ile Ile Tyr Asn Glu Trp Gln Met Asn Asp Ile Lys Pro Leu Lys Phe340 345 350Lys Ile Tyr His Gly Gln355<210>111<211>358<212>PRT<213>金黄色葡萄球菌(Staphylococcus aureus)<400>111Met Ile Gln Val Lys Ala Pro Gly Lys Leu Tyr Ile Ala Gly Glu Tyr1 5 10 15Ala Val Thr Glu Pro Gly Tyr Lys Ser Val Leu Ile Ala Leu Asp Arg20 25 30Phe Val Thr Ala Thr Ile Glu Glu Ala Thr Gln Tyr Lys Gly Thr Ile35 40 45His Ser Lys Ala Leu His His Asn Pro Val Thr Phe Ser Arg Asp Glu50 55 60Asp Ser Ile Val Ile Ser Asp Pro His Ala Ala Lys Gln Leu Asn Tyr65 70 75 80Val Val Thr Ala Ile Glu Ile Phe Glu Gln Tyr Ala Lys Ser Cys Asp85 90 95Ile Ala Met Lys His Phe His Leu Thr Ile Asp Ser Asn Leu Asp Asp100 105 110Ser Asn Gly His Lys Tyr Gly Leu Gly Ser Ser Ala Ala Val Leu Val115 120 125Ser Val Ile Lys Val Leu Asn Glu Phe Tyr Asp Met Lys Leu Ser Asn130 135 140Leu Tyr Ile Tyr Lys Leu Ala Val Ile Ala Asn Met Lys Leu Gln Ser145 150 155 160Leu Ser Ser Cys Gly Asp Ile Ala Val Ser Val Tyr Ser Gly Trp Leu165 170 175
Ala Tyr Ser Thr Phe Asp His Glu Trp Val Lys His Gln Ile Glu Asp180 185 190Thr Thr Val Glu Glu Val Leu Ile Lys Asn Trp Pro Gly Leu His Ile195 200 205Glu Pro Leu Gln Ala Pro Glu Asn Met Glu Val Leu Ile Gly Trp Thr210 215 220Gly Ser Pro Ala Ser Ser Pro His Phe Val Ser Glu Val Lys Arg Leu225 230 235 240Lys Ser Asp Pro Ser Phe Tyr Gly Asp Phe Leu Glu Asp Ser His Arg245 250 255Cys Val Glu Lys Leu Ile His Ala Phe Lys Thr Asn Asn Ile Lys Gly260 265 270Val Gln Lys Met Val Arg Gln Asn Arg Thr Ile Ile Gln Arg Met Asp275 280 285Lys Glu Ala Thr Val Asp Ile Glu Thr Glu Lys Leu Lys Tyr Leu Cys290 295 300Asp Ile Ala Glu Lys Tyr His Gly Ala Ser Lys Thr Ser Gly Ala Gly305 310 315 320Gly Gly Asp Cys Gly Ile Thr Ile Ile Asn Lys Asp Val Asp Lys Glu325 330 335Lys Ile Tyr Asp Glu Trp Thr Lys His Gly Ile Lys Pro Leu Lys Phe340 345 350Asn Ile Tyr His Gly Gln355<210>112<211>374<212>PRT<213>链霉菌属菌种(Streptomyces sp.)<400>112Met Thr Thr Gly Gln Arg Thr Ile Val Arg His Ala Pro Gly Lys Leu1 5 10 15Phe Val Ala Gly Glu Tyr Ala Val Val Asp Pro Gly Asn Pro Ala Ile20 25 30Leu Val Ala Val Asp Arg His Ile Ser Val Thr Val Ser Asp Ala Asp35 40 45Ala Asp Thr Gly Ala Ala Asp Val Val Ile Ser Ser Asp Leu Gly Pro50 55 60Gln Ala Val Gly Trp Arg Trp His Asp Gly Arg Leu Val Val Arg Asp65 70 75 80Pro Asp Asp Gly Gln Gln Ala Arg Ser Ala Leu Ala His Val Val Ser85 90 95Ala Ile Glu Thr Val Gly Arg Leu Leu Gly Glu Arg Gly Gln Lys Val100 105 110
Pro Ala Leu Thr Leu Ser Val Ser Ser Arg Leu His Glu Asp Gly Arg115 120 125Lys Phe Gly Leu Gly Ser Ser Gly Ala Val Thr Val Ala Thr Val Ala130 135 140Ala Val Ala Ala Phe Cys Gly Leu Glu Leu Ser Thr Asp Glu Arg Phe145 150 155 160Arg Leu Ala Met Leu Ala Thr Ala Glu Leu Asp Pro Lys Gly Ser Gly165 170 175Gly Asp Leu Ala Ala Ser Thr Trp Gly Gly Trp Ile Ala Tyr Gln Ala180 185 190Pro Asp Arg Ala Phe Val Leu Asp Leu Ala Arg Arg Val Gly Val Asp195 200 205Arg Thr Leu Lys Ala Pro Trp Pro Gly His Ser Val Arg Arg Leu Pro210 215 220Ala Pro Lys Gly Leu Thr Leu Glu Val Gly Trp Thr Gly Glu Pro Ala225 230 235 240Ser Thr Ala Ser Leu Val Ser Asp Leu His Arg Arg Thr Trp Arg Gly245 250 255Ser Ala Ser His Gln Arg Phe Val Glu Thr Thr Thr Asp Cys Val Arg260 265 270Ser Ala Val Thr Ala Leu Glu Ser Gly Asp Asp Thr Ser Leu Leu His275 280 285Glu Ile Arg Arg Ala Arg Gln Glu Leu Ala Arg Leu Asp Asp Glu Val290 295 300Gly Leu Gly Ile Phe Thr Pro Lys Leu Thr Ala Leu Cys Asp Ala Ala305 310 315 320Glu Ala Val Gly Gly Ala Ala Lys Pro Ser Gly Ala Gly Gly Gly Asp325 330 335Cys Gly Ile Ala Leu Leu Asp Ala Glu Ala Ser Arg Asp Ile Thr His340 345 350Val Arg Gln Arg Trp Glu Thr Ala Gly Val Leu Pro Leu Pro Leu Thr355 360 365Pro Ala Leu Glu Gly Ile370<210>113<211>360<212>PRT<213>灰孢链霉菌(Streptomyces griseosporeus)<400>113Met Thr Gly Pro Arg Ala Val Thr Arg Arg Ala Pro Gly Lys Leu Phe1 5 10 15
Val Ala Gly Glu Tyr Ala Val Val Glu Pro Gly Asn Arg Ala Ile Leu20 25 30Val Ala Val Asp Arg Tyr Val Thr Val Thr Val Ser Asp Gly Ala Ala35 40 45Pro Gly Val Val Val Ser Ser Asp Ile Gly Ala Gly Pro Val His His50 55 60Pro Trp Gln Asp Gly Arg Leu Thr Gly Gly Thr Gly Thr Pro His Val65 70 75 80Val Ala Ala Val Glu Thr Val Ala Arg Leu Leu Ala Glu Arg Gly Arg85 90 95Ser Val Pro Pro Leu Gly Trp Ser Ile Ser Ser Thr Leu His Glu Asp100 105 110Gly Arg Lys Phe Gly Leu Gly Ser Ser Gly Ala Val Thr Val Ala Thr115 120 125Val Ser Ala Val Ala Ala His Cys Gly Leu Glu Leu Thr Ala Glu Glu130 135 140Arg Phe Arg Thr Ala Leu Ile Ala Ser Ala Arg Ile Asp Pro Arg Gly145 150 155 160Ser Gly Gly Asp Ile Ala Thr Ser Thr Trp Gly Gly Trp Ile Ala Tyr165 170 175Arg Ala Pro Asp Arg Asp Ala Val Leu Asp Leu Thr Arg Arg Gln Gly180 185 190Val Asp Glu Ala Leu Arg Ala Pro Trp Pro Gly Phe Ser Val Arg Leu195 200 205Ser Pro Pro Arg Asn Leu Cys Leu Glu Val Gly Trp Thr Gly Asn Pro210 215 220Val Ser Thr Thr Ser Leu Leu Thr Asp Leu His Arg Arg Thr Trp Arg225 230 235 240Gly Ser Pro Ala Tyr Arg Arg Tyr Val Gly Ala Thr Gly Glu Leu Val245 250 255Asp Ala Ala Val Ile Ala Leu Glu Asp Gly Asp Thr Glu Gly Leu Leu260 265 270Arg Gln Val Arg Arg Ala Arg His Glu Met Val Arg Leu Asp Asp Glu275 280 285Val Gly Leu Gly Ile Phe Thr Pro Glu Leu Thr Ala Leu Cys Ala Ile290 295 300Ala Glu Arg Ala Gly Ala Ala Lys Pro Ser Gly Ala Gly Gly Gly Asp305 310 315 320Cys Gly Ile Ala Leu Leu Asp Ala Glu Ala Arg Tyr Asp Arg Ser Pro325 330 335
Leu His Arg Gln Trp Ala Ala Ala Gly Val Leu Pro Leu Leu Val Ser340 345 350Pro Ala Thr Glu Gly Val Glu Glu355 360<210>114<211>317<212>PRT<213>布氏疏螺旋体(Borrelia burgdorferi)<400>114Met Asp Leu Ile Ser Phe Ser Val Pro Gly Asn Leu Leu Leu Met Gly1 5 10 15Glu Tyr Thr Ile Leu Glu Glu Lys Gly Leu Gly Leu Ala Ile Ala Ile20 25 30Asn Lys Arg Ala Phe Phe Ser Phe Lys Lys Ser Asp Ser Trp Arg Phe35 40 45Phe Ser Lys Lys Lys Lys Ile Asp Asp Phe Ser Leu Ile Glu Asn Arg50 55 60Ser Asp Phe Val Phe Lys Met Phe Ala Tyr Leu Ser Gln Asn Cys Phe65 70 75 80Phe Asn Leu Glu Asn Phe Ala Tyr Asp Val Tyr Ile Asp Thr Ser Asn85 90 95Phe Phe Phe Asn Asp Gly Thr Lys Lys Gly Phe Gly Ser Ser Ala Val100 105 110Val Ala Ile Gly Ile Val Cys Gly Leu Phe Leu Ile His Asn Ala Thr115 120 125Asn Val Val Glu Lys Gly Glu Ile Phe Lys Tyr Cys Leu Glu Ala Tyr130 135 140Arg Tyr Ser Gln Gly Gly Ile Gly Ser Gly Tyr Asp Ile Ala Thr Ser145 150 155 160Ile Phe Gly Gly Val Ile Glu Phe Glu Gly Gly Phe Asn Pro Lys Cys165 170 175Arg Gln Leu Gly Ala Val Glu Phe Asn Asp Phe Tyr Leu Met Gln Gly180 185 190Leu Gln Ala Ile Lys Thr Thr Thr Ser Ile Cys Glu Tyr Asn Lys His195 200 205Arg Asn Ser Ile Leu Asp Phe Ile Leu Lys Cys Asn Leu Glu Met Lys210 215 220Lys Leu Val Leu Asn Ala Ser Asn Ser Lys Ser Ala Leu Ile Ser Ser225 230 235 240Leu Arg Arg Ala Lys Glu Leu Gly Leu Ala Ile Gly Glu Ala Ile Gly245 250 255Val Ser Ala Ala Leu Pro Ser Ser Phe Asp His Leu Leu Gly Gln Cys260 265 270
Asp Leu Ile Lys Ala Leu Gly Ala Gly Asn Glu Thr Phe Leu Val Tyr275 280 285Arg Pro Asn Ile Glu Ala Phe Asn Leu Ser Lys Ile Ile Ser Ile Val290 295 300Leu Glu Asn Glu Gly Ile Lys Phe Glu Ser Asp Lys Cys305 310 315<210>115<211>30<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(30)<223>引物<400>115gggcaagctt gtccacggca cgaccaagca 30<210>116<211>30<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(30)<223>引物<400>116cgtaatccgc ggccgcgttt ccagcgcgtc 30<210>117<211>28<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(28)<223>引物<400>117aattaaagga gggtttcata tgaattcg 28<210>118<211>28<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(28)<223>引物<400>118gatccgaatt catatgaaac cctccttt 28<210>119<211>30<212>DNA<213>人工的<220>
<221>misc_feature
<222>(1)..(30)<223>引物<400>119aaggcctcat atgatttccc ataccccggt 30<210>120<211>28<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(28)<223>引物<400>120cgggatcctc atcgctccat ctccatgt 28<210>121<211>30<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(30)<223>引物<400>121aaggcctcat atgaccgaca gcaaggatca 30<210>122<211>28<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(28)<223>引物<400>122cgggatcctc attgacggat aagcgagg 28<210>123<211>29<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(29)<223>引物<400>123aaggcctcat atgaaagtgc ctaagatga29<210>124<211>28<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(28)<223>引物<400>124cgggatcctc aggcctgccg gtcgacat 28<210>125<211>34
<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(34)<223>引物<400>125aaggcctcat atgagcaccg gcaggcctga agca 34<210>126<211>31<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(31)<223>引物<400>126cgggatcctc atccctgccc cggcagcggt t31<210>127<211>30<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(30)<223>引物<400>127aaggcctcat atggatcagg tcatccgcgc 30<210>128<211>28<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(28)<223>引物<400>128cgggatcctc agtcatcgaa aacaagtc 28<210>129<211>30<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(30)<223>引物<400>129aaggcctcat atgactgatg ccgtccgcga 30<210>130<211>28<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(28)<223>引物
<400>130cgggatcctc aacgcccctc gaacggcg 28<210>131<211>31<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(31)<223>引物<400>131ccggcattcg ggcggcatcc aggtctcgct g 31<210>132<211>31<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(31)<223>引物<400>132cagcgagacc tggatgccgc ccgaatgccg g 31<210>133<211>31<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(31)<223>引物<400>133cgtgcagggc tggattctgt cggaataccc g 31<210>134<211>31<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(31)<223>引物<400>134cgggtattcc gacagaatcc agccctgcac g 31<210>135<211>31<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(31)<223>引物<400>135gggctgcgcg ccggcatccg gcatttcgac g 31<210>136<211>31<212>DNA<213>人工的
<220>
<221>misc_feature<222>(1)..(31)<223>引物<400>136cgtcgaaatg ccggatgccg gcgcgcagcc c 31<210>137<211>31<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(31)<223>引物<400>137gggtgcgacg ggcgagttct tcgatgcgcg g 31<210>138<211>31<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(31)<223>引物<400>138ccgcgcatcg aagaactcgc ccgtcgcacc c 31<210>139<211>31<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(31)<223>引物<400>139cacgcccgtc acatacgacg aatacgttgc c 31<210>140<211>31<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(31)<223>引物<400>140ggcaacgtat tcgtcgtatg tgacgggcgt g 31<210>141<211>31<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(31)<223>引物<400>141gaggctcggg cttggctcct cggcggcggt g 31
<210>142<211>31<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(31)<223>引物<400>142caccgccgcc gaggagccaa gcccgagcct c 31<210>143<211>31<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(31)<223>引物<400>143cggcacgctg ctggacccgg gcgacgcctt c31<210>144<211>31<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(31)<223>引物<400>144gaaggcgtcg cccgggtcca gcagcgtgcc g 31<210>145<211>36<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(36)<223>寡核苷酸<400>145tcagaattcg gtaccatatg aagcttggat ccgggg 36<210>146<211>29<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(29)<223>寡核苷酸<400>146ggatccaagc ttcatatggt accgaattc 29<210>147<211>26<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(26)<223>引物<400>147ggaattcgct gctgaacgcg atggcg 26<210>148<211>32<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(32)<223>引物<400>148ggggtaccat atgtgccttc gttgcgtcag tc32<210>149<211>50<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(50)<223>寡核苷酸<400>149gatccggcgt gtgcgcaatt taattgcgca cacgccccct gcgtttaaac 50<210>150<211>50<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(50)<223>寡核苷酸<400>150gatcgtttaa acgcaggggg cgtgtgcgca attaaattgc gcacacgccg 50<210>151<211>30<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(30)<223>引物<400>151aaggcctcat atgacgccca agcagcaatt 30<210>152<211>26<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(26)<223>引物<400>152cgggatccta ggcgctgcgg cggatg 26
<210>153<211>30<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(30)<223>引物<400>153ccggatcctc atgcctgccg gtcgacatag 30<210>154<211>30<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(30)<223>引物<400>154gaaggcacat atgaatcagg tcatccgcgc 30<210>155<211>30<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(30)<223>引物<400>155gccggatcct cattcatcga aaacaagtcc 30<210>156<211>30<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(30)<223>引物<400>156acgccggatc ctcatcgccc ctcgaacggc 30<210>157<211>1612<212>DNA<213>副球菌属菌种(Paracoccus sp.)<220>
<221>基因<222>(1)..(1612)<223>
<400>157ccatggcatc cgggtcggat gccgtctatg ttggcccgaa caggcagcag gaggccccat 60gagcgatatc cagaccctct cgttcgagga agccatgcgc gagctggagg cgaccgtcgg 120caagctggaa accggcgagg cgacgctcga ggactccatc gcgctctatg aacgcggggc 180ggcgctgcgc gcccattgcg aaacccgcct gcgcgaggcc gaggagcggg tcgagaagat 240caccctggcc gcgaacgggc agccgtccgg aaccgagccc gccgagggcc tgtgatgcag 300gcccgcctgg ccgagatccg gcccctggtc gaggccgagc tgaacgccgc catcgacgcg 360ctgcccgcgg gcgatctgtc ggatgcgatg cgctatgccg tgcagggcgg caagcggctg 420cgcgcgttcc tggtgatgga gtcggcgcgc ctgcacgggc tggacgacga cgcatcgctg 480
cccgtcgccg ccgcggtcga ggcgctgcac gcctacagct tggtccatga cgacctgccc 540gcgatggatg acgacgacct gcggcgcggt cagcccaccg tccacgtcaa atggaccgag 600gcgaccgcga tccttgcggg cgatgcgctg cagacgctgg ccttccagct gctggccgat 660ccgcgcgtgg gcgacgatgc ggcgcggatg cggctggtcg gttcgctggc gcaggcatcg 720ggggctgcgg gcatggtctg gggccaggcg ctggacatcg cggccgagac ctcgggcgtg 780ccgctggatc tggacgcgat catccgcctg cagggtggca agaccggcgc gctgatccgc 840tttgccgcga ccgccgggcc gctgatggcg ggggcggacc ctgccgcgct ggacgattat 900gcgcaggccg tcgggctggc cttccagatc gcggacgaca tcctggacgt cgagggctgc 960gaggccgcga ccggcaagcg cgtcggcaag gatgcggatg ccaacaaggc gaccttcgtc1020tcgctgctgg gcctcgaggg ggcgcggtcc gaggcgcgtc gcctggccga tgcggggcag1080gacgcgctgg cgggttacgg cgatgctgcg gggaaccttc gggacctggc gcgcttcgtg1140atcgaacgcg acagctgatc gccgccttcc cgccaagggg caagatgatg accgacggac1200ccgcaacccc gatcctggac cgcgtccagc agccatccga cctggcatcg ctggacgatg1260cgcagctgcg cctgctggcg gacgagctgc gggccgagac catcgacatc gtcagccgca1320cgggcggtca cctgggcgcg gggctgggcg tggtcgaact gacggtcgcc ctgcacgccg1380tctttcgggc gccgcgcgac aagatcgtct gggacgtggg gcatcaatgc tatccccaca1440agatcctgac gggcaggcgg gaccggatgc gcacgctgcg catgggcggc gggctgtcgg1500ggttcaccaa gcggcaggaa agcgcgttcg atccgttcgg tgcggggcac agctcgacct1560cgatctcggc ggcgctgggc ttcgcgatgg cgcgtgaact tggcggggat cc1612<210>158<211>78<212>PRT<213>副球菌属菌种(Paracoccus sp.)<400>158Met Ser Asp Ile Gln Thr Leu Ser Phe Glu Glu Ala Met Arg Glu Leu1 5 10 15Glu Ala Thr Val Gly Lys Leu Glu Thr Gly Glu Ala Thr Leu Glu Asp20 25 30Ser Ile Ala Leu Tyr Glu Arg Gly Ala Ala Leu Arg Ala His Cys Glu35 40 45Thr Arg Leu Arg Glu Ala Glu Glu Arg Val Glu Lys Ile Thr Leu Ala50 55 60Ala Asn Gly Gln Pro Ser Gly Thr Glu Pro Ala Glu Gly Leu65 70 75<210>159<211>287<212>PRT<213>副球菌属菌种(Paracoccus sp.)<400>159Met Gln Ala Arg Leu Ala Glu Ile Arg Pro Leu Val Glu Ala Glu Leu1 5 10 15Asn Ala Ala Ile Asp Ala Leu Pro Ala Gly Asp Leu Ser Asp Ala Met20 25 30Arg Tyr Ala Val Gln Gly Gly Lys Arg Leu Arg Ala Phe Leu Val Met35 40 45Glu Ser Ala Arg Leu His Gly Leu Asp Asp Asp Ala Ser Leu Pro Val50 55 60Ala Ala Ala Val Glu Ala Leu His Ala Tyr Ser Leu Val His Asp Asp65 70 75 80
Leu Pro Ala Met Asp Asp Asp Asp Leu Arg Arg Gly Gln Pro Thr Val85 90 95His Val Lys Trp Thr Glu Ala Thr Ala Ile Leu Ala Gly Asp Ala Leu100 105 110Gln Thr Leu Ala Phe Gln Leu Leu Ala Asp Pro Arg Val Gly Asp Asp115 120 125Ala Ala Arg Met Arg Leu Val Gly Ser Leu Ala Gln Ala Ser Gly Ala130 135 140Ala Gly Met Val Trp Gly Gln Ala Leu Asp Ile Ala Ala Glu Thr Ser145 150 155 160Gly Val Pro Leu Asp Leu Asp Ala Ile Ile Arg Leu Gln Gly Gly Lys165 170 175Thr Gly Ala Leu Ile Arg Phe Ala Ala Thr Ala Gly Pro Leu Met Ala180 185 190Gly Ala Asp Pro Ala Ala Leu Asp Asp Tyr Ala Gln Ala Val Gly Leu195 200 205Ala Phe Gln Ile Ala Asp Asp Ile Leu Asp Val Glu Gly Cys Glu Ala210 215 220Ala Thr Gly Lys Arg Val Gly Lys Asp Ala Asp Ala Asn Lys Ala Thr225 230 235 240Phe Val Ser Leu Leu Gly Leu Glu Gly Ala Arg Ser Glu Ala Arg Arg245 250 255Leu Ala Asp Ala Gly Gln Asp Ala Leu Ala Gly Tyr Gly Asp Ala Ala260 265 270Gly Asn Leu Arg Asp Leu Ala Arg Phe Val Ile Glu Arg Asp Ser275 280 285<210>160<211>142<212>PRT<213>副球菌属菌种(Paracoccus sp.)<400>160Met Met Thr Asp Gly Pro Ala Thr Pro Ile Leu Asp Arg Val Gln Gln1 5 10 15Pro Ser Asp Leu Ala Ser Leu Asp Asp Ala Gln Leu Arg Leu Leu Ala20 25 30Asp Glu Leu Arg Ala Glu Thr Ile Asp Ile Val Ser Arg Thr Gly Gly35 40 45His Leu Gly Ala Gly Leu Gly Val Val Glu Leu Thr Val Ala Leu His50 55 60Ala Val Phe Arg Ala Pro Arg Asp Lys Ile Val Trp Asp Val Gly His65 70 75 80
Gln Cys Tyr Pro His Lys Ile Leu Thr Gly Arg Arg Asp Arg Met Arg85 90 95Thr Leu Arg Met Gly Gly Gly Leu Ser Gly Phe Thr Lys Arg Gln Glu100 105 110Ser Ala Phe Asp Pro Phe Gly Ala Gly His Ser Ser Thr Ser Ile Ser115 120 125Ala Ala Leu Gly Phe Ala Met Ala Arg Glu Leu Gly Gly Asp130 135 140<210>161<211>6<212>PRT<213>大豆慢生根瘤菌(Bradyrhizobium japonicum)<400>161Val His Asp Asp Leu Pro1 5<210>162<211>6<212>PRT<213>根瘤菌属菌种(Rhizobium sp.)<400>162Val His Asp Asp Leu Pro1 5<210>163<211>6<212>PRT<213>嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)<400>163Ile His Asp Asp Leu Pro1 5<210>164<211>6<212>PRT<213>枯草芽孢杆菌(Bacillus subtilis)<400>164Ile His Asp Asp Leu Pro1 5<210>165<211>6<212>PRT<213>大肠杆菌(Escherichia coli)<400>165Ile His Asp Asp Leu Pro1 5<210>166<211>6<212>PRT<213>流感嗜血菌Haemophilus influenzae)<400>166Ile His Asp Asp Leu Pro1 5
<210>167<211>16<212>DNA<213>人工的<220>
<221>misc_feature<222>(5)..(5)<223>n代表C或T<220>
<221>misc_feature<222>(8)..(8)<223>n代表C或T<220>
<221>misc_feature<222>(11)..(11)<223>n代表C或T<220>
<221>misc_feature<222>(1)..(16)<223>寡核苷酸<400>167tccanganga nctgcc 16<210>168<211>5<212>PRT<213>大豆慢生根瘤菌(Bradyrhizobium japonicum)<400>168Asp Asp Ile Leu Asp1 5<210>169<211>5<212>PRT<213>根瘤菌属菌种(Rhizobium sp.)<400>169Asp Asp Ile Leu Asp1 5<210>170<211>5<212>PRT<213>嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)<400>170Asp Asp Ile Leu Asp1 5<210>171<211>5<212>PRT<213>枯草芽孢杆菌(Bacillus subtilis)<400>171Asp Asp Ile Leu Asp1 5<210>172<211>5<212>PRT
<213>大肠杆菌(Escherichia coli)<400>172Asp Asp Ile Leu Asp1 5<210>173<211>5<212>PRT<213>流感嗜血菌Haemophilus influenzae)<400>173Asp Asp Ile Leu Asp1 5<210>174<211>15<212>DNA<213>人工的<220>
<221>misc_feature<222>(3)..(3)<223>n代表C或T<220>
<221>misc_feature<222>(6)..(6)<223>n代表C或T<220>
<221>misc_feature<222>(15)..(15)<223>n代表C或T<220>
<221>misc_feature<222>(1)..(15)<223>寡核苷酸<400>174ganganatcc tggan15<210>175<211>1176<212>DNA<213>副球菌属菌种(Paracoccus sp.)<220>
<221>CDS<222>(1)..(1173)<223>
<400>175atg gac ccc atc gtc atc acc ggc gcg atg cgc acc ccg atg ggg gca 48Met Asp Pro Ile Val Ile Thr Gly Ala Met Arg Thr Pro Met Gly Ala1 5 10 15ttc cag ggc gat ctt gcc gcg atg gat gcc ccg acc ctt ggc gcg gcc 96Phe Gln Gly Asp Leu Ala Ala Met Asp Ala Pro Thr Leu Gly Ala Ala20 25 30gcg atc cgc gcc gcg ctg aac ggc ctg tcg ccc gac atg gtg gac gag144Ala Ile Arg Ala Ala Leu Asn Gly Leu Ser Pro Asp Met Val Asp Glu35 40 45gtg ctg atg ggc tgc gtc ctg ccc gcg ggc cag ggt cag gca ccg gca192Val Leu Met Gly Cys Val Leu Pro Ala Gly Gln Gly Gln Ala Pro Ala50 55 60
cgt cag gcg gcg ctt gac gcc gga ctg ccg ctg tcg gcg ggc gcg acc 240Arg Gln Ala Ala Leu Asp Ala Gly Leu Pro Leu Ser Ala Gly Ala Thr65 70 75 80acc atc aac aag atg tgc gga tcg ggc atg aag gcc gcg atg ctg ggc 288Thr Ile Asn Lys Met Cys Gly Ser Gly Met Lys Ala Ala Met Leu Gly85 90 95cat gac ctg atc gcc gcg gga tcg gcg ggc atc gtc gtc gcc ggc ggg 336His Asp Leu Ile Ala Ala Gly Ser Ala Gly Ile Val Val Ala Gly Gly100 105 110atg gag agc atg tcg aac gcc ccc tac ctg ctg ccc aag gcg cgg tcg 384Met Glu Ser Met Ser Asn Ala Pro Tyr Leu Leu Pro Lys Ala Arg Ser115 120 125ggg atg cgc atg ggc cat gac cgt gtg ctg gat cac atg ttc ctc gac 432Gly Met Arg Met Gly His Asp Arg Val Leu Asp His Met Phe Leu Asp130 135 140ggg ttg gag gac gcc tat gac aag ggc cgc ctg atg ggc acc ttc gcc 480Gly Leu Glu Asp Ala Tyr Asp Lys Gly Arg Leu Met Gly Thr Phe Ala145 150 155 160gag gat tgc gcc ggc gat cac ggt ttc acc cgc gag gcg cag gac gac 528Glu Asp Cys Ala Gly Asp His Gly Phe Thr Arg Glu Ala Gln Asp Asp165 170 175tat gcg ctg acc agc ctg gcc cgc gcg cag gac gcc atc gcc agc ggt 576Tyr Ala Leu Thr Ser Leu Ala Arg Ala Gln Asp Ala Ile Ala Ser Gly180 185 190gcc ttc gcc gcc gag atc gcg ccc gtg acc gtc acg gca cgc aag gtg 624Ala Phe Ala Ala Glu Ile Ala Pro Val Thr Val Thr Ala Arg Lys Val195 200 205cag acc acc gtc gat acc gac gag atg ccc ggc aag gcc cgc ccc gag 672Gln Thr Thr Val Asp Thr Asp Glu Met Pro Gly Lys Ala Arg Pro Glu210 215 220aag atc ccc cat ctg aag ccc gcc ttc cgt gac ggt ggc acg gtc acg 720Lys Ile Pro His Leu Lys Pro Ala Phe Arg Asp Gly Gly Thr Val Thr225 230 235 240gcg gcg aac agc tcg tcg atc tcg gac ggg gcg gcg gcg ctg gtg atg 768Ala Ala Asn Ser Ser Ser Ile Ser Asp Gly Ala Ala Ala Leu Val Met245 250 255atg cgc cag tcg cag gcc gag aag ctg ggc ctg acg ccg atc gcg cgg 816Met Arg Gln Ser Gln Ala Glu Lys Leu Gly Leu Thr Pro Ile Ala Arg260 265 270atc atc ggt cat gcg acc cat gcc gac cgt ccc ggc ctg ttc ccg acg 864Ile Ile Gly His Ala Thr His Ala Asp Arg Pro Gly Leu Phe Pro Thr275 280 285gcc ccc atc ggc gcg atg cgc aag ctg ctg gac cgc acg gac acc cgc 912Ala Pro Ile Gly Ala Met Arg Lys Leu Leu Asp Arg Thr Asp Thr Arg290 295 300
ctt ggc gat tac gac ctg ttc gag gtg aac gag gca ttc gcc gtc gtc 960Leu Gly Asp Tyr Asp Leu Phe Glu Val Asn Glu Ala Phe Ala Val Val305 310 315 320gcc atg atc gcg atg aag gag ctt ggc ctg cca cac gat gcc acg aac1008Ala Met Ile Ala Met Lys Glu Leu Gly Leu Pro His Asp Ala Thr Asn325 330 335atc aac ggc ggg gcc tgc gcg ctt ggg cat ccc atc ggc gcg tcg ggg1056Ile Asn Gly Gly Ala Cys Ala Leu Gly His Pro Ile Gly Ala Ser Gly340 345 350gcg cgg atc atg gtc acg ctg ctg aac gcg atg gcg gcg cgg ggc gcg1104Ala Arg Ile Met Val Thr Leu Leu Asn Ala Met Ala Ala Arg Gly Ala355 360 365acg cgc ggg gcc gca tcc gtc tgc atc ggc ggg ggc gag gcg acg gcc1152Thr Arg Gly Ala Ala Ser Val Cys Ile Gly Gly Gly Glu Ala Thr Ala370 375 380atc gcg ctg gaa cgg ctg agc taa1176Ile Ala Leu Glu Arg Leu Ser385 390<210>176<211>391<212>PRT<213>副球菌属菌种(Paracoccus sp.)<400>176Met Asp Pro Ile Val Ile Thr Gly Ala Met Arg Thr Pro Met Gly Ala1 5 10 15Phe Gln Gly Asp Leu Ala Ala Met Asp Ala Pro Thr Leu Gly Ala Ala20 25 30Ala Ile Arg Ala Ala Leu Asn Gly Leu Ser Pro Asp Met Val Asp Glu35 40 45Val Leu Met Gly Cys Val Leu Pro Ala Gly Gln Gly Gln Ala Pro Ala50 55 60Arg Gln Ala Ala Leu Asp Ala Gly Leu Pro Leu Ser Ala Gly Ala Thr65 70 75 80Thr Ile Asn Lys Met Cys Gly Ser Gly Met Lys Ala Ala Met Leu Gly85 90 95His Asp Leu Ile Ala Ala Gly Ser Ala Gly Ile Val Val Ala Gly Gly100 105 110Met Glu Ser Met Ser Asn Ala Pro Tyr Leu Leu Pro Lys Ala Arg Ser115 120 125Gly Met Arg Met Gly His Asp Arg Val Leu Asp His Met Phe Leu Asp130 135 140Gly Leu Glu Asp Ala Tyr Asp Lys Gly Arg Leu Met Gly Thr Phe Ala145 150 155 160
Glu Asp Cys Ala Gly Asp His Gly Phe Thr Arg Glu Ala Gln Asp Asp165 170 175Tyr Ala Leu Thr Ser Leu Ala Arg Ala Gln Asp Ala Ile Ala Ser Gly180 185 190Ala Phe Ala Ala Glu Ile Ala Pro Val Thr Val Thr Ala Arg Lys Val195 200 205Gln Thr Thr Val Asp Thr Asp Glu Met Pro Gly Lys Ala Arg Pro Glu210 215 220Lys Ile Pro His Leu Lys Pro Ala Phe Arg Asp Gly Gly Thr Val Thr225 230 235 240Ala Ala Asn Ser Ser Ser Ile Ser Asp Gly Ala Ala Ala Leu Val Met245 250 255Met Arg Gln Ser Gln Ala Glu Lys Leu Gly Leu Thr Pro Ile Ala Arg260 265 270Ile Ile Gly His Ala Thr His Ala Asp Arg Pro Gly Leu Phe Pro Thr275 280 285Ala Pro Ile Gly Ala Met Arg Lys Leu Leu Asp Arg Thr Asp Thr Arg290 295 300Leu Gly Asp Tyr Asp Leu Phe Glu Val Asn Glu Ala Phe Ala Val Val305 310 315 320Ala Met Ile Ala Met Lys Glu Leu Gly Leu Pro His Asp Ala Thr Asn325 330 335Ile Asn Gly Gly Ala Cys Ala Leu Gly His Pro Ile Gly Ala Ser Gly340 345 350Ala Arg Ile Met Val Thr Leu Leu Asn Ala Met Ala Ala Arg Gly Ala355 360 365Thr Arg Gly Ala Ala Ser Val Cys Ile Gly Gly Gly Glu Ala Thr Ala370 375 380Ile Ala Leu Glu Arg Leu Ser385 390<210>177<211>1980<212>DNA<213>副球菌属菌种(Paracoccus sp.)<220>
<221>基因<222>(1)..(1980)<223>
<400>177atgaccaaag ccgtaatcgt atctgccgca cgtacccccg tcggcagctt catgggcgca 60ttcgccaatg tccccgcaca tgatctgggc gccgccgtcc tgcgcgaggt cgtggcccgc 120gccggtgtcg accccgccga ggtcagcgag acgatcctgg gccaggtgct gaccgccgcg 180cagggccaga accccgcgcg ccaggcgcat atcaatgcgg gcctgcccaa ggaatcggcg 240
gcgtggctca tcaaccaggt ctgcggctcg gggctgcgcg ccgtcgcgct ggcggcgcag 300caggtcatgc tgggcgatgc gcagatcgtt ctggcggggg gccaggagag catgtcgctg 360tcgacccatg ccgcctatct gcgcgcgggc cagaagatgg gcgacatgaa gatgatcgac 420accatgatcc gcgacgggct gtgggatgcc ttcaacggct atcacatggg tcagaccgcc 480gagaacgtgg ccgaccagtg gtcgatcagc cgcgaccagc aggacgaatt cgccctggct 540tcgcagaaca aggccgaggc cgcgcagaat gcgggccgct tcgatgacga aatcgtcgcc 600tataccgtca agggccgcaa gggcgacacg gtcgtcgaca aggacgaata catccgccac 660ggcgccacga tcgagggcat gcagaagctg cgccccgcct tcaccaagga aggctcggtc 720acggcgggca acgcgtcggg cctgaacgac ggcgcggcgg ccgtcatggt catgtccgag 780gacgaggccg cacgccgcgg gctgacgccg ctggcgcgca tcgcctccta tgcgacggcg 840ggcctcgacc cggcgatcat gggcaccggg ccgatcccct ccagccgcaa ggcgctggaa 900aaggcgggct ggtcggtcgg cgacctggac ctggtcgagg cgaacgaggc ctttgccgcg 960caggcctgcg ccgtgaacaa ggacatgggc tgggatccgt ccatcgtgaa cgtcaacggc1020ggcgcgatcg ccatcggcca cccgatcggc gcctcggggg cgcggatcct gaacaccctg1080ctgttcgaga tgcagcgccg cgacgccaag aagggccttg cgacgctgtg catcggcggc1140ggcatgggcg tcgccatgtg cctcgaacgc tgaacgaccg gcgtgtgcgc aatttaattg1200cgcacacgcc ccctgcaaag tagcaatgtt ttacgataac gaatgaaggg gggaatcatg1260tccaaggtag cactggtcac cggcggatcg cgcggcatcg gcgccgagat ctgcaaggcg1320cttcaggccg caggctatac cgtcgccgcg aactatgccg gcaatgacga cgcggccaag1380gccttcaccg aggaaaccgg catcaagacc tacaagtggt cggtcgccga ttacgatgcc1440tgcaaggccg gcatcgccca ggtcgaagag gatctgggcc cgatcgccgt gctgatcaac1500aatgccggga tcacccgcga cgcgcccttc cacaagatga cgcccgagaa gtggaaggag1560gtcatcgaca ccaacctgac cggcaccttc aacatgaccc atccggtctg gccgggcatg1620cgcgaacgca agttcggacg cgtcatcaac atcagctcga tcaacgggca gaagggccag1680ttcgggcagg cgaactatgc cgcggccaag gcgggcgacc tgggcttcac caagtcgctg1740gcgcaggaag gcgcgcgcaa caacatcacc gtcaacgcga tctgccccgg ctatatcgcg1800acggacatgg tgatggccgt tcccgaacag gtccgcgagg ggatcatcgc gcagatcccc1860gtcggccgct tgggcgagcc gtccgagatc gcgcgctgcg tggtgttcct ggcctccgac1920gatgcgggct tcgtcacagg ctcgaccatc acggcgaatg gcggccagta ctacatctga1980<210>178<211>390<212>PRT<213>副球菌属菌种(Paracoccus sp.)<400>178Met Thr Lys Ala Val Ile Val Ser Ala Ala Arg Thr Pro Val Gly Ser1 5 10 15Phe Met Gly Ala Phe Ala Asn Val Pro Ala His Asp Leu Gly Ala Ala20 25 30Val Leu Arg Glu Val Val Ala Arg Ala Gly Val Asp Pro Ala Glu Val35 40 45Ser Glu Thr Ile Leu Gly Gln Val Leu Thr Ala Ala Gln Gly Gln Asn50 55 60Pro Ala Arg Gln Ala His Ile Asn Ala Gly Leu Pro Lys Glu Ser Ala65 70 75 80Ala Trp Leu Ile Asn Gln Val Cys Gly Ser Gly Leu Arg Ala Val Ala85 90 95Leu Ala Ala Gln Gln Val Met Leu Gly Asp Ala Gln Ile Val Leu Ala100 105 110Gly Gly Gln Glu Ser Met Ser Leu Ser Thr His Ala Ala Tyr Leu Arg115 120 125Ala Gly Gln Lys Met Gly Asp Met Lys Met Ile Asp Thr Met Ile Arg130 135 140
Asp Gly Leu Trp Asp Ala Phe Asn Gly Tyr His Met Gly Gln Thr Ala145 150 155 160Glu Asn Val Ala Asp Gln Trp Ser Ile Ser Arg Asp Gln Gln Asp Glu165 170 175Phe Ala Leu Ala Ser Gln Asn Lys Ala Glu Ala Ala Gln Asn Ala Gly180 185 190Arg Phe Asp Asp Glu Ile Val Ala Tyr Thr Val Lys Gly Arg Lys Gly195 200 205Asp Thr Val Val Asp Lys Asp Glu Tyr Ile Arg His Gly Ala Thr Ile210 215 220Glu Gly Met Gln Lys Leu Arg Pro Ala Phe Thr Lys Glu Gly Ser Val225 230 235 240Thr Ala Gly Asn Ala Ser Gly Leu Asn Asp Gly Ala Ala Ala Val Met245 250 255Val Met Ser Glu Asp Glu Ala Ala Arg Arg Gly Leu Thr Pro Leu Ala260 265 270Arg Ile Ala Ser Tyr Ala Thr Ala Gly Leu Asp Pro Ala Ile Met Gly275 280 285Thr Gly Pro Ile Pro Ser Ser Arg Lys Ala Leu Glu Lys Ala Gly Trp290 295 300Ser Val Gly Asp Leu Asp Leu Val Glu Ala Asn Glu Ala Phe Ala Ala305 310 315 320Gln Ala Cys Ala Val Asn Lys Asp Met Gly Trp Asp Pro Ser Ile Val325 330 335Asn Val Asn Gly Gly Ala Ile Ala Ile Gly His Pro Ile Gly Ala Ser340 345 350Gly Ala Arg Ile Leu Asn Thr Leu Leu Phe Glu Met Gln Arg Arg Asp355 360 365Ala Lys Lys Gly Leu Ala Thr Leu Cys Ile Gly Gly Gly Met Gly Val370 375 380Ala Met Cys Leu Glu Arg385 390<210>179<211>240<212>PRT<213>副球菌属菌种(Paracoccus sp.)<400>179Met Ser Lys Val Ala Leu Val Thr Gly Gly Ser Arg Gly Ile Gly Ala1 5 10 15Glu Ile Cys Lys Ala Leu Gln Ala Ala Gly Tyr Thr Val Ala Ala Asn20 25 30
Tyr Ala Gly Asn Asp Asp Ala Ala Lys Ala Phe Thr Glu Glu Thr Gly35 40 45Ile Lys Thr Tyr Lys Trp Ser Val Ala Asp Tyr Asp Ala Cys Lys Ala50 55 60Gly Ile Ala Gln Val Glu Glu Asp Leu Gly Pro Ile Ala Val Leu Ile65 70 75 80Asn Asn Ala Gly Ile Thr Arg Asp Ala Pro Phe His Lys Met Thr Pro85 90 95Glu Lys Trp Lys Glu Val Ile Asp Thr Asn Leu Thr Gly Thr Phe Asn100 105 110Met Thr His Pro Val Trp Pro Gly Met Arg Glu Arg Lys Phe Gly Arg115 120 125Val Ile Asn Ile Ser Ser Ile Asn Gly Gln Lys Gly Gln Phe Gly Gln130 135 140Ala Asn Tyr Ala Ala Ala Lys Ala Gly Asp Leu Gly Phe Thr Lys Ser145 150 155 160Leu Ala Gln Glu Gly Ala Arg Asn Asn Ile Thr Val Asn Ala Ile Cys165 170 175Pro Gly Tyr Ile Ala Thr Asp Met Val Met Ala Val Pro Glu Gln Val180 185 190Arg Glu Gly Ile Ile Ala Gln Ile Pro Val Gly Arg Leu Gly Glu Pro195 200 205Ser Glu Ile Ala Arg Cys Val Val Phe Leu Ala Ser Asp Asp Ala Gly210 215 220Phe Val Thr Gly Ser Thr Ile Thr Ala Asn Gly Gly Gln Tyr Tyr Ile225 230 235 240<210>180<211>729<212>DNA<213>Paracoccus carotinifaciens E-396<220>
<221>CDS<222>(1)..(726)<223>
<400>180atg agc gca cat gcc ctg ccc aag gca gat ctg acc gcc acc agt ttg 48Met Ser Ala His Ala Leu Pro Lys Ala Asp Leu Thr Ala Thr Ser Leu1 5 10 15atc gtc tcg ggc ggc atc atc gcc gcg tgg ctg gcc ctg cat gtg cat 96Ile Val Ser Gly Gly Ile Ile Ala Ala Trp Leu Ala Leu His Val His20 25 30gcg ctg tgg ttt ctg gac gcg gcg gcg cat ccc atc ctg gcg gtc gcg144Ala Leu Trp Phe Leu Asp Ala Ala Ala His Pro Ile Leu Ala Val Ala
35 40 45aat ttc ctg ggg ctg acc tgg ctg tcg gtc ggt ctg ttc atc atc gcg192Asn Phe Leu Gly Leu Thr Trp Leu Ser Val Gly Leu Phe Ile Ile Ala50 55 60cat gac gcg atg cat ggg tcg gtc gtg ccg ggg cgc ccg cgc gcc aat240His Asp Ala Met His Gly Ser Val Val Pro Gly Arg Pro Arg Ala Asn65 70 75 80gcg gcg atg ggc cag ctt gtc ctg tgg ctg tat gcc gga ttt tcc tgg288Ala Ala Met Gly Gln Leu Val Leu Trp Leu Tyr Ala Gly Phe Ser Trp85 90 95cgc aag atg atc gtc aag cac atg gcc cat cat cgc cat gcc gga acc336Arg Lys Met Ile Val Lys His Met Ala His His Arg His Ala Gly Thr100 105 110gac gac gac cca gat ttc gac cat ggc ggc ccg gtc cgc tgg tac gcc384Asp Asp Asp Pro Asp Phe Asp His Gly Gly Pro Val Arg Trp Tyr Ala115 120 125cgc ttc atc ggc acc tat ttc ggc tgg cgc gag ggg ctg ctg ctg ccc432Arg Phe Ile Gly Thr Tyr Phe Gly Trp Arg Glu Gly Leu Leu Leu Pro130 135 140gtc atc gtg acg gtc tat gcg ctg atg ttg ggg gat cgc tgg atg tac480Val Ile Val Thr Val Tyr Ala Leu Met Leu Gly Asp Arg Trp Met Tyr145 150 155 160gtg gtc ttc tgg ccg ttg ccg tcg atc ctg gcg tcg atc cag ctg ttc528Val Val Phe Trp Pro Leu Pro Ser Ile Leu Ala Ser Ile Gln Leu Phe165 170 175gtg ttc ggc atc tgg ctg ccg cac cgc ccc ggc cac gac gcg ttc ccg576Val Phe Gly Ile Trp Leu Pro His Arg Pro Gly His Asp Ala Phe Pro180 185 190gac cgc cac aat gcg cgg tcg tcg cgg atc agc gac ccc gtg tcg ctg624Asp Arg His Asn Ala Arg Ser Ser Arg Ile Ser Asp Pro Val Ser Leu195 200 205ctg acc tgc ttt cac ttt ggc ggt tat cat cac gaa cac cac ctg cac672Leu Thr Cys Phe His Phe Gly Gly Tyr His His Glu His His Leu His210 215 220ccg acg gtg cct tgg tgg cgc ctg ccc agc acc cgc acc aag ggg gac720Pro Thr Val Pro Trp Trp Arg Leu Pro Ser Thr Arg Thr Lys Gly Asp225 230 235 240acc gca tga729Thr Ala<210>181<211>242<212>PRT<213>Paracoccus carotinifaciens E-396<400>181Met Ser Ala His Ala Leu Pro Lys Ala Asp Leu Thr Ala Thr Ser Leu1 5 10 15
Ile Val Ser Gly Gly Ile Ile Ala Ala Trp Leu Ala Leu His Val His20 25 30Ala Leu Trp Phe Leu Asp Ala Ala Ala His Pro Ile Leu Ala Val Ala35 40 45Asn Phe Leu Gly Leu Thr Trp Leu Ser Val Gly Leu Phe Ile Ile Ala50 55 60His Asp Ala Met His Gly Ser Val Val Pro Gly Arg Pro Arg Ala Asn65 70 75 80Ala Ala Met Gly Gln Leu Val Leu Trp Leu Tyr Ala Gly Phe Ser Trp85 90 95Arg Lys Met Ile Val Lys His Met Ala His His Arg His Ala Gly Thr100 105 110Asp Asp Asp Pro Asp Phe Asp His Gly Gly Pro Val Arg Trp Tyr Ala115 120 125Arg Phe Ile Gly Thr Tyr Phe Gly Trp Arg Glu Gly Leu Leu Leu Pro130 135 140Val Ile Val Thr Val Tyr Ala Leu Met Leu Gly Asp Arg Trp Met Tyr145 150 155 160Val Val Phe Trp Pro Leu Pro Ser Ile Leu Ala Ser Ile Gln Leu Phe165 170 175Val Phe Gly Ile Trp Leu Pro His Arg Pro Gly His Asp Ala Phe Pro180 185 190Asp Arg His Asn Ala Arg Ser Ser Arg Ile Ser Asp Pro Val Ser Leu195 200 205Leu Thr Cys Phe His Phe Gly Gly Tyr His His Glu His His Leu His210 215 220Pro Thr Val Pro Trp Trp Arg Leu Pro Ser Thr Arg Thr Lys Gly Asp225 230 235 240Thr Ala<210>182<211>510<212>DNA<213>副球菌属菌种(Paracoccus sp.)<220>
<221>CDS<222>(1)..(507)<223>
<400>182atg agc act tgg gcc gca atc ctg acc gtc atc ctg acc gtc gcc gcg48Met Ser Thr Trp Ala Ala Ile Leu Thr Val Ile Leu Thr Val Ala Ala1 5 10 15atg gag ctg acg gcc tac tcc gtc cat cgg tgg atc atg cat ggc ccc96Met Glu Leu Thr Ala Tyr Ser Val His Arg Trp Ile Met His Gly Pro
20 25 30ctg ggc tgg ggc tgg cat aaa tcg cac cac gac gag gat cac gac cac144Leu Gly Trp Gly Trp His Lys Ser His His Asp Glu Asp His Asp His35 40 45gcg ctc gag aag aac gac ctc tat ggc gtc atc ttc gcg gta atc tcg192Ala Leu Glu Lys Asn Asp Leu Tyr Gly Val Ile Phe Ala Val Ile Ser50 55 60atc gtg ctg ttc gcg atc ggc gcg atg ggg tcg gat ctg gcc tgg tgg240Ile Val Leu Phe Ala Ile Gly Ala Met Gly Ser Asp Leu Ala Trp Trp65 70 75 80ctg gcg gtg ggg gtc acc tgc tac ggg ctg atc tac tat ttc ctg cat288Leu Ala Val Gly Val Thr Cys Tyr Gly Leu Ile Tyr Tyr Phe Leu His85 90 95gac ggc ttg gtg cat ggg cgc tgg ccg ttc cgc tat gtc ccc aag cgc336Asp Gly Leu Val His Gly Arg Trp Pro Phe Arg Tyr Val Pro Lys Arg100 105 110ggc tat ctt cgt cgc gtc tac cag gca cac agg atg cat cac gcg gtc384Gly Tyr Leu Arg Arg Val Tyr Gln Ala His Arg Met His His Ala Val115 120 125cat ggc cgc gag aac tgc gtc agc ttc ggt ttc atc tgg gcg ccc tcg432His Gly Arg Glu Asn Cys Val Ser Phe Gly Phe Ile Trp Ala Pro Ser130 135 140gtc gac agc ctc aag gca gag ctg aaa cgc tcg ggc gcg ctg ctg aag480Val Asp Ser Leu Lys Ala Glu Leu Lys Arg Ser Gly Ala Leu Leu Lys145 150 155 160gac cgc gaa ggg gcg gat cgc aat aca tga510Asp Arg Glu Gly Ala Asp Arg Asn Thr165<210>183<211>169<212>PRT<213>副球菌属菌种(Paracoccus sp.)<400>183Met Ser Thr Trp Ala Ala Ile Leu Thr Val Ile Leu Thr Val Ala Ala1 5 10 15Met Glu Leu Thr Ala Tyr Ser Val His Arg Trp Ile Met His Gly Pro20 25 30Leu Gly Trp Gly Trp His Lys Ser His His Asp Glu Asp His Asp His35 40 45Ala Leu Glu Lys Asn Asp Leu Tyr Gly Val Ile Phe Ala Val Ile Ser50 55 60Ile Val Leu Phe Ala Ile Gly Ala Met Gly Ser Asp Leu Ala Trp Trp65 70 75 80Leu Ala Val Gly Val Thr Cys Tyr Gly Leu Ile Tyr Tyr Phe Leu His
85 90 95Asp Gly Leu Val His Gly Arg Trp Pro Phe Arg Tyr Val Pro Lys Arg100 105 110Gly Tyr Leu Arg Arg Val Tyr Gln Ala His Arg Met His His Ala Val115 120 125His Gly Arg Glu Asn Cys Val Ser Phe Gly Phe Ile Trp Ala Pro Ser130 135 140Val Asp Ser Leu Lys Ala Glu Leu Lys Arg Ser Gly Ala Leu Leu Lys145 150 155 160Asp Arg Glu Gly Ala Asp Arg Asn Thr165<210>184<211>888<212>DNA<213>副球菌属菌种(Paracoccus sp.)<220>
<221>CDS<222>(1)..(885)<223>
<400>184atg acg ccc aag cag caa ttc ccc cta cgc gat ctg gtc gag atc agg 48Met Thr Pro Lys Gln Gln Phe Pro Leu Arg Asp Leu Val Glu Ile Arg1 5 10 15ctg gcg cag atc tcg ggc cag ttc ggc gtg gtc tcg gcc ccg ctc ggc 96Leu Ala Gln Ile Ser Gly Gln Phe Gly Val Val Ser Ala Pro Leu Gly20 25 30gcg gcc atg agc gat gcc gcc ctg tcc ccc ggc aaa cgc ttt cgc gcc144Ala Ala Met Ser Asp Ala Ala Leu Ser Pro Gly Lys Arg Phe Arg Ala35 40 45gtg ctg atg ctg atg gtc gcc gaa agc tcg ggc ggg gtc tgc gat gcg192Val Leu Met Leu Met Val Ala Glu Ser Ser Gly Gly Val Cys Asp Ala50 55 60atg gtc gat gcc gcc tgc gcg gtc gag atg gtc cat gcc gca tcg ctg240Met Val Asp Ala Ala Cys Ala Val Glu Met Val His Ala Ala Ser Leu65 70 75 80atc ttc gac gac atg ccc tgc atg gac gat gcc agg acc cgt cgc ggt288Ile Phe Asp Asp Met Pro Cys Met Asp Asp Ala Arg Thr Arg Arg Gly85 90 95cag ccc gcc acc cat gtc gcc cat ggc gag ggg cgc gcg gtg ctt gcg336Gln Pro Ala Thr His Val Ala His Gly Glu Gly Arg Ala Val Leu Ala100 105 110ggc atc gcc ctg atc acc gag gcc atg cgg att ttg ggc gag gcg cgc384Gly Ile Ala Leu Ile Thr Glu Ala Met Arg Ile Leu Gly Glu Ala Arg115 120 125ggc gcg acg ccg gat cag cgc gca agg ctg gtc gca tcc atg tcg cgc432Gly Ala Thr Pro Asp Gln Arg Ala Arg Leu Val Ala Ser Met Ser Arg130 135 140
gcg atg gga ccg gtg ggg ctg tgc gca ggg cag gat ctg gac ctg cac480Ala Met Gly Pro Val Gly Leu Cys Ala Gly Gln Asp Leu Asp Leu His145 150 155 160gcc ccc aag gac gcc gcc ggg atc gaa cgt gaa cag gac ctc aag acc528Ala Pro Lys Asp Ala Ala Gly Ile Glu Arg Glu Gln Asp Leu Lys Thr165 170 175ggc gtg ctg ttc gtc gcg ggc ctc gag atg ctg tcc att att aag ggt576Gly Val Leu Phe Val Ala Gly Leu Glu Met Leu Ser Ile Ile Lys Gly180 185 190ctg gac aag gcc gag acc gag cag ctc atg gcc ttc ggg cgt cag ctt624Leu Asp Lys Ala Glu Thr Glu Gln Leu Met Ala Phe Gly Arg Gln Leu195 200 205ggt cgg gtc ttc cag tcc tat gac gac ctg ctg gac gtg atc ggc gac672Gly Arg Val Phe Gln Ser Tyr Asp Asp Leu Leu Asp Val Ile Gly Asp210 215 220aag gcc agc acc ggc aag gat acg ggg cgc gac acc gcc gcc ccc ggc720Lys Ala Ser Thr Gly Lys Asp Thr Gly Arg Asp Thr Ala Ala Pro Gly225 230 235 240cca aag cgc ggc ctg atg gcg gtc gga cag atg ggc gac gtg gcg cag768Pro Lys Arg Gly Leu Met Ala Val Gly Gln Met Gly Asp Val Ala Gln245 250 255cat tac cgc gcc agc cgc gcg caa ctg gac gag ctg atg cgc acc cgg816His Tyr Arg Ala Ser Arg Ala Gln Leu Asp Glu Leu Met Arg Thr Arg260 265 270ctg ttc cgc ggg ggg cag atc gcg gac ctg ctg gcc cgc gtg ctg ccg864Leu Phe Arg Gly Gly Gln Ile Ala Asp Leu Leu Ala Arg Val Leu Pro275 280 285cat gac atc cgc cgc agc gcc tag888His Asp Ile Arg Arg Ser Ala290 295<210>185<211>295<212>PRT<213>副球菌属菌种(Paracoccus sp.)<400>185Met Thr Pro Lys Gln Gln Phe Pro Leu Arg Asp Leu Val Glu Ile Arg1 5 10 15Leu Ala Gln Ile Ser Gly Gln Phe Gly Val Val Ser Ala Pro Leu Gly20 25 30Ala Ala Met Ser Asp Ala Ala Leu Ser Pro Gly Lys Arg Phe Arg Ala35 40 45Val Leu Met Leu Met Val Ala Glu Ser Ser Gly Gly Val Cys Asp Ala50 55 60Met Val Asp Ala Ala Cys Ala Val Glu Met Val His Ala Ala Ser Leu65 70 75 80Ile Phe Asp Asp Met Pro Cys Met Asp Asp Ala Arg Thr Arg Arg Gly
85 90 95Gln Pro Ala Thr His Val Ala His Gly Glu Gly Arg Ala Val Leu Ala100 105 110Gly Ile Ala Leu Ile Thr Glu Ala Met Arg Ile Leu Gly Glu Ala Arg115 120 125Gly Ala Thr Pro Asp Gln Arg Ala Arg Leu Val Ala Ser Met Ser Arg130 135 140Ala Met Gly Pro Val Gly Leu Cys Ala Gly Gln Asp Leu Asp Leu His145 150 155 160Ala Pro Lys Asp Ala Ala Gly Ile Glu Arg Glu Gln Asp Leu Lys Thr165 170 175Gly Val Leu Phe Val Ala Gly Leu Glu Met Leu Ser Ile Ile Lys Gly180 185 190Leu Asp Lys Ala Glu Thr Glu Gln Leu Met Ala Phe Gly Arg Gln Leu195 200 205Gly Arg Val Phe Gln Ser Tyr Asp Asp Leu Leu Asp Val Ile Gly Asp210 215 220Lys Ala Ser Thr Gly Lys Asp Thr Gly Arg Asp Thr Ala Ala Pro Gly225 230 235 240Pro Lys Arg Gly Leu Met Ala Val Gly Gln Met Gly Asp Val Ala Gln245 250 255His Tyr Arg Ala Ser Arg Ala Gln Leu Asp Glu Leu Met Arg Thr Arg260 265 270Leu Phe Arg Gly Gly Gln Ile Ala Asp Leu Leu Ala Arg Val Leu Pro275 280 285His Asp Ile Arg Arg Ser Ala290 295<210>186<211>30<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(30)<223>引物<400>186aaggcctcat atgagcgcac atgccctgcc 30<210>187<211>28<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(28)<223>引物
<400>187cgggatcctc atgcggtgtc ccccttgg 28<210>188<211>30<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(30)<223>引物<400>188aaggcctcat atgagcactt gggccgcaat 30<210>189<211>30<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(30)<223>引物<400>189aggatcctca tgtattgcga tccgcccctt 30<210>190<211>52<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(52)<223>多接头<400>190gtgcagcctc aggtcgacat atgcggccgc atccggatcc ctcctcctcc ag 52<210>191<211>52<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(52)<223>多接头<400>191cacgtcggag tccagctgta tacgccggcg taggcctagg gaggaggagg tc 52<210>192<211>52<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(52)<223>多接头<400>192gtgcaggagg aggtcgacat atgcggccgc atccggatcc ctgaggctcc ag 52<210>193<211>52<212>DNA
<213>人工的<220>
<221>misc_feature<222>(1)..(52)<223>多接头<400>193cacgtcctcc tccagctgta tacgccggcg taggcctagg gactccgagg tc 52<210>194<211>52<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(52)<223>多接头<400>194ctggagcctc aggtcgacat atgcggccgc atccggatcc ctcctcctgc ac 52<210>195<211>52<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(52)<223>多接头<400>195gacctcggag tccagctgta tacgccggcg taggcctagg gaggaggacg tg 52<210>196<211>52<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(52)<223>多接头<400>196ctggaggagg aggtcgacat atgcggccgc atccggatcc ctgaggctgc ac 52<210>197<211>52<212>DNA<213>人工的<220>
<221>misc_feature<222>(1)..(52)<223>多接头<400>197gacctcctcc tccagctgta tacgccggcg taggcctagg gactccgacg tg 5权利要求
1.分离的多肽,其包括选自下述的氨基酸序列(a)SEQ ID NO43的1-340残基所示的氨基酸序列;(b)SEQ ID NO45的1-349残基所示的氨基酸序列;(c)SEQ ID NO47的1-388残基所示的氨基酸序列;(d)SEQ ID NO49的1-378残基所示的氨基酸序列;(e)SEQ ID NO51的1-305残基所示的氨基酸序列;(f)SEQ ID NO53的1-332残基所示的氨基酸序列;(g)选自SEQ ID NO43,45,47,49,51或53所示氨基酸序列的片段,其中所述片段至少具有30个连续的氨基酸残基;(h)选自SEQ ID NO43,45,47,49,51或53所示多肽的片段的氨基酸序列,该片段具有羟甲戊二酰-CoA还原酶(HMG-CoA还原酶),异戊烯二磷酸异构酶,羟甲戊二酰-CoA合成酶(HMG-CoA合成酶),甲羟戊酸激酶,磷酸甲羟戊酸激酶,或二磷酸甲羟戊酸脱羧酶的活性;(i)严谨条件下能够与杂交探针杂交的多核苷酸所编码的多肽的氨基酸序列,所述杂交探针含有SEQ ID NO42或SEQ ID NO42的互补序列的至少30个连续核苷酸,其中所述多肽具有HMG-CoA还原酶,异戊烯二磷酸异构酶,HMG-CoA合成酶,甲羟戊酸激酶,磷酸甲羟戊酸激酶,或二磷酸甲羟戊酸脱羧酶的活性;和(j)SEQ ID NO43,45,47,49,51或53的保守修饰的变体。
2.分离的多肽,其包括选自下述的氨基酸序列(a)SEQ ID NO159的1-287残基所示氨基酸序列;(b)SEQ ID NO159的至少30个连续的氨基酸残基;(c)SEQ ID NO159的片段的氨基酸序列,所述片段具有法呢基二磷酸合成酶(FPP合成酶)的活性;(d)在严谨条件下能够与杂交探针杂交的多核苷酸所编码的多肽的氨基酸序列,所述杂交探针含有跨SEQ ID NO157的295-1158位核苷酸或其互补序列的至少30个连续核苷酸,其中所述多肽具有FPP合成酶的活性;和(e)SEQ ID NO159的保守修饰的变体。
3.分离的多肽,其含有选自下述的氨基酸序列(a)SEQ ID NO160的残基1-142所示的氨基酸序列;(b)SEQ ID NO160的至少30个连续的氨基酸残基;(c)SEQ ID NO160的片段的氨基酸序列,所述片段具有1-脱氧木酮糖-5-磷酸合成酶(DXPS)的活性;(d)在严谨条件下能够与杂交探针杂交的多核苷酸所编码的多肽的氨基酸序列,所述杂交探针含有SEQ ID NO157的1185-1610位核苷酸或其互补序列的至少30个连续核苷酸,其中所述多肽具有DXPS的活性;(e)SEQ ID NO160的保守修饰的变体。
4.分离的多肽,其含有选自下述的氨基酸序列(a)SEQ ID NO178的1-390残基所示的氨基酸序列;(b)SEQ ID NO178的至少30个连续氨基酸残基;(c)SEQ ID NO178的多肽片段的氨基酸序列,所述片段具有乙酰-CoA乙酰基转移酶的活性;(d)在严谨条件下能够与杂交探针杂交的多核苷酸所编码的多肽的氨基酸序列,所述杂交探针含有SEQ ID NO177的1-1170位核苷酸或其互补序列的至少30个连续核苷酸,其中所述多肽具有乙酰-CoA乙酰基转移酶的活性;和(e)SEQ ID NO178保守修饰的变体。
5.分离的多肽,其含有选自下述的氨基酸序列(a)SEQ ID NO179的1-240残基所示的氨基酸序列;(b)SEQ ID NO179的至少30个连续的氨基酸残基;(c)SEQ ID NO179的多肽片段的氨基酸序列,所述片段具有乙酰乙酰-CoA还原酶的活性;(d)在严谨条件下能够与杂交探针杂交的多核苷酸所编码的多肽氨基酸序列,所述杂交探针含有SEQ ID NO177或其互补序列的1258-1980位核苷酸的至少30个连续核苷酸,其中所述多肽具有乙酰乙酰-CoA还原酶的活性;和(e)SEQ ID NO179保守修饰的变体。
6.分离的多核苷酸序列,其含有选自下述的核苷酸序列SEQ ID NO42;SEQ ID NO42的变体,该变体含有一个或多个根据副球菌属的菌株R1534密码子使用表的取代;SEQ ID NO42的片段,该片段编码具有选自下述活性的多肽羟甲戊二酰-CoA还原酶(HMG-CoA还原酶),异戊烯二磷酸异构酶,羟甲戊二酰-CoA合成酶(HMG-CoA合成酶),甲羟戊酸激酶,磷酸甲羟戊酸激酶,或二磷酸甲羟戊酸脱羧酶;或在严谨条件下能够与杂交探针杂交的多核苷酸序列,该探针的核苷酸序列由SEQ ID NO42或SEQ IDNO42的互补序列的至少30个连续核苷酸构成,所述的多核苷酸编码具有选自下述活性的多肽HMG-CoA还原酶,异戊烯二磷酸异构酶,HMG-CoA合成酶,甲羟戊酸激酶,磷酸甲羟戊酸激酶或二磷酸甲羟戊酸脱羧酶。
7.分离的多核苷酸序列,其含有选自下述的多核苷酸序列SEQ ID NO157的核苷酸序列;SEQ ID NO157变体,该变体含有一个或多个根据副球菌属的菌株R1534密码子使用表的取代;SEQ ID NO157的片段,该片段编码具有下述活性的多肽法呢基二磷酸合成酶(FPP合成酶)活性,1-脱氧-D-木酮糖-5-磷酸合成酶活性或具有XseB活性的多肽;或在严谨条件下能够与杂交探针杂交的多核苷酸序列,该探针的核苷酸序列由SEQ ID NO157或SEQ ID NO157的互补序列的至少30个连续核苷酸构成,所述的多核苷酸编码具有下述活性的多肽FPP合成酶活性,1-脱氧-D-木酮糖-5-磷酸合成酶活性或XseB活性。
8.分离的多核苷酸序列,其含有选自下述的多核苷酸序列SEQ ID NO177的核苷酸序列;SEQ ID NO177的变体;该变体含有一个或多个根据副球菌属的菌株R1534密码子使用表的取代;SEQ ID NO177的片段,该片段编码具有选自乙酰-CoA乙酰基转移酶活性或乙酰乙酰-CoA还原酶活性的多肽;或在严谨条件下能够与杂交探针杂交的多核苷酸序列,该探针的核苷酸序列由SEQ ID NO177或SEQ ID NO177的互补序列的至少30个连续核苷酸构成,所述的多核苷酸编码具有选自乙酰-CoA乙酰基转移酶活性或乙酰乙酰-CoA还原酶活性的多肽。
9.分离的多核苷酸序列,其包括选自下述的核苷酸序列SEQ ID NO42,SEQ ID NO157,SEQ ID NO177或其组合。
10.含有权利要求6,7,8,或9的多核苷酸序列的表达载体。
11.选自下述的表达载体pBBR-K-mev-op16-1,pBBR-K-mev-op16-2,pDS-mvaA,pDS-idi,pDS-hcs,pDS-mvk,pDS-pmk,pDS-mvd,pDS-His-mvaA,pDS-His-idi,pDS-His-hcs,pDS-His-mvk,pDS-His-pmk,pDS-His-mvd,pBBR-K-Zea4,pBBR-K-Zea4-up,pBBR-K-Zea4-down,pBBR-K-PcrtE-crtE-3,pBBR-tK-PcrtE-mvaA,pBBR-tK-PcrtE-idi,pBBR-tK-PcrtE-hcs,pBBR-tK-PcrtE-mvk,pBBR-tK-PcrtE-pmk,pBBR-tK-PcrtE-mvd,pBBR-K-PcrtE-mvaA-crtE-3,pDS-His-phaA,pBBR-K-PcrtE-crtW,pBBR-K-PcrtE-crtWZ,pBBR-K-PcrtE-crtZW或其组合。
12.培养的细胞或该细胞的后代,其中所述细胞含有权利要求6,7,8或9的多核苷酸序列或权利要求10或11的表达载体,其中所述细胞表达由所述多核苷酸序列所编码的多肽。
13.生产类胡萝卜素的方法,包括在允许表达所述多核苷酸序列编码的多肽的条件下,培养权利要求12的细胞,并从细胞或者该细胞培养基中分离类胡萝卜素。
14.制备产类胡萝卜素细胞的方法,包括(a)将编码甲羟戊酸途径中的酶的多核苷酸序列导入细胞,所述酶在该细胞中能够表达;和(b)选择出含有步骤(a)多核苷酸序列的细胞,并且该细胞所生产类胡萝卜素的水平为该细胞导入多核苷酸序列之前的约1.1-1000倍。
15.一种改造细菌以使其生产类异戊二烯化合物的方法,包括(a)在允许表达类异戊二烯化合物的条件下,在培养基中培养亲代细菌,并从该培养基中筛选出突变细菌,所述突变细菌生产的类异戊二烯化合物比亲代细菌高约1.1-1000倍;(b)将含有SEQ ID NO42多核苷酸序列的表达载体导入突变细菌,所述多核苷酸序列与表达调控序列可操作连接;和(c)筛选出含有表达载体并且生产的类异戊二烯化合物比步骤(a)中的突变体高出至少约1.1倍的细菌。
16.副球菌属的微生物,所述微生物具有下述特征(i)使用GeneCompar v.2.0软件,缺口罚分为0%的条件下,利用同源性计算获得的相似性的矩阵,与SEQ ID NO12的序列相似性>97%;在81.5℃,使用DNA:DNA杂交,与菌株R-1512,R1534,R114或R-1506的同源性>70%;其基因组DNA的G+C含量与R114,R-1512,R1534和R-1506的基因组DNA的G+C含量相比,变化小于1%;和使用实施例2的AFLP方法,平均DNA指纹与菌株R-1512,R1534,R114和R-1506具有约58%的相似性,附带条件为该微生物不是副球菌属菌种(MBIC3966);(ii)含有至少约75%的细胞膜总脂肪酸的18:1w7c;不具有使用福寿草醇,i-赤藓糖醇,龙胆二糖,β-甲基葡糖苷,D-山梨糖醇,木糖醇和奎尼酸作为碳源用于生长的能力;和能够使用L-天冬酰胺和L-天冬氨酸作为碳源用于生长,附带条件为该微生物不是副球菌属菌种(MBIC3966);或(iii)能够在40℃生长;能够在含有8%NaCl的培养基中生长;能够在pH9.1的培养基中生长;和形成黄色-橙色色素的菌落,附带条件为该微生物不是副球菌属菌种(MBIC3966)。
全文摘要
提供了分离的多核苷酸,其编码具有甲羟戊酸途径各酶活性的多肽,所述酶例如,甲羟戊二酸单酰-CoA还原酶,异戊烯二磷酸异构酶,甲羟戊二酸单酰-CoA合成酶,甲羟戊酸激酶,磷酸甲羟戊酸激酶,或二磷酸甲羟戊酸脱羧酶,该多核苷酸可用于重组生产类异戊二烯化合物,如类胡萝卜素,例如八氢番茄红素,番茄红素,β-胡萝卜素,玉米黄质,角黄素,虾青素,福寿草黄素(adonixanthin),隐黄素,海胆酮和福寿草红素(adonirubin)。还提供了表达载体,培养的细胞,以及生产类异戊二烯化合物的方法。
文档编号C12N9/90GK1630718SQ02811380
公开日2005年6月22日 申请日期2002年6月5日 优先权日2001年6月6日
发明者艾伦·贝里, 沃纳·布雷策尔, 马库斯·许布林, 鲁阿尔·洛佩兹-尤利巴里, 安妮·F·迈耶, 亚历克西·耶利西夫 申请人:Dsm Ip资产公司
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